Antisense design

ABSTRACT

A novel class of pharmaceuticals which comprises a Locked Nucleic Acid (LNA) which can be used in antisense therapy. These novel oligonucleotides have improved antisense properties. The novel oligonucleotides are composed of at least one LNA selected from beta-D-thio/amino-LNA or alpha-L-oxy/thio/amino-LNA. The oligonucleotides comprising LNA may also include DNA and/or RNA nucleotides.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 15/237,307, whichwas filed Aug. 15, 2016, which is a continuation of U.S. Ser. No.13/797,919, which was filed on Mar. 12, 2013, which claims priority toU.S. application Ser. No. 10/535,472, which was filed on Dec. 19, 2005,which is a National Phase Application of PCT/DK2003/00788, which wasfiled Nov. 18, 2003 and Application No. PA2002/01774, filed Nov. 18,2002, and Application No. PA2003/01540, filed Oct. 20, 2003.

FIELD OF INVENTION

The present invention relates to pharmaceuticals comprising antisenseoligonucleotides, and novel oligonucleotides having improved antisenseproperties.

BACKGROUND OF THE INVENTION

The Professors Imanishi and Wengel independently invented Locked NucleicAcid (LNA) in 1997 (International Patent Applications WO 99/14226,WO98/39352; P. Nielsen et al, J. Chem. Soc., Perkin Trans. 1, 1997, 3423;P. Nielsen et al., Chem. Commun., 1997, 9, 825; N. K. Christensen etal., J. Am. Chem. Soc., 1998, 120, 5458; A. A. Koshkin et al., J. Org.Chem., 1998, 63, 2778; A. A Koshkin et al. J. Am. Chem. Soc. 1998, 120,13252-53; Kumar et al. Bioorg, & Med. Chem. Lett.,1998, 8, 2219-2222;and S. Obika et al., Bioorg. Med. Chem. Lett., 1999, 515). The first LNAmonomer was based on the 2′-O—CH2-4′ bicyclic structure. Due to theconfiguration of this structure it is called: beta-D-oxy-LNA. Thisoxy-LNA has since then showed promising biological applications (Braasch& Corey, Biochemistry, 2002, 41(14), 4503-19; Childs et al. PNAS, 2002,99(17), 11091-96; Crinelli et al., Nucl. Acid. Res., 2002, 30(11),2435-43; Elayadi et al., Biochemistry, 2002, 41, 25 9973-9981; Jacobsenet al., Nucl. Acid. Res., 2002, 30(19), in press; Kurreck et al., Nucl.Acid. Res., 2002, 30(9),1911-1918; Simeonov & Nikiforov, Nucl. Acid.Res., 2002, 30(17); Alayadi & Corey, Curr. opinion in Inves. Drugs.,2001, 2(4), 558-61; Obika et al., Bioorg. & Med. Chem., 2001, 9,1001-11; Braasch & Corey, Chem. & Biol., 2000, 55, 1-7; Wahlestedt etal., PNAS, 2000, 97(10), 5633-38), Freier & Altmann, Nucl. Acid Res.,1997, 25, 4429-43; Cook, 1999, Nucleosides & Nucleotides, 18(6&7),1141-62.

Right after the discovery of oxy-LNA the bicyclic furanosidic structurewas chemically derivatised. Thus, the 2′-S—CH₂-4′ (thio-LNA) and the2′-NH—CH₂-4′ (amino-LNA) bicyclic analogues were disclosed (Singh, S.K., J. Org. Chem., 1998, 63, 6078-79; Kumar et al. Bioorg, & Med. Chem.Lett.,1998, 8, 2219-2222; Singh et al. J. Org. Chem., 1998, 63,10035-39). The synthesis of the thio-LNA containing uridine asnucleobase has been shown (Singh, S. K., J. Org. Chem., 1998, 63,6078-79). For amino-LNA the synthesis of the thymidine nucleobase hasbeen disclosed (Kumar et al. Bioorg, & Med. Chem. Lett.,1998, 8,2219-2222; Singh et al. J. Org. Chem., 1998, 63, 10035-39). A series ofLNA-diastereoisomers have been prepared (Rajwanshi et al., J. ChemCommun. 1999;2073-2074; Hakansson & Wengel, Bioorg Med Chem Lett 2001;11(7):935-938; Rajwanshi et al., Chem Commun., 1999;1395-1396; Wengel atal., Nucleosides Nucleotides Nucleic Acids, 2001; 20(4-7):389-396;Rajwanshi et al., Angew. Chem. Int. Ed., 2000; 39:1656-1659; Petersen etal., J. Amer. Chem. Soc., 2001, 123(30), 7431-32; Sorensen et al., J.Amer. Chem. Soc., 2002, 124(10), 2164-76; Vester et al., J. Amer. Chem.Soc., 2002, 124(46), 13682-13683). In the prior art the synthesis ofalpha-L-xylo, xylo-LNA, and alpha-L-oxy-LNA containing thymidine baseshave been shown. For the alpha-L-oxy-LNA also the 5-methyl and adeninenucleosides have been synthesised. The melting temperature (Tm) ofduplexes containing the LNA distereoisomers have been presented. Itturned out that the alpha-L-oxy-LNA has interesting properties. It wasshown that the alpha-L-oxy-LNA can be incorporated in complex chimeraecomprising DNA/RNA residues and be adapted in the oligo structure andincrease the binding. This property of being incorporated inoligonucleotides containing several other monomeric classes and actco-operatively is a property that the alpha-L-oxy-LNA shares with theparent oxy-LNA. Furthermore, it has been demonstrated that a segment of4 consecutive alpha-L-T monomers can be incorporated in conjunction witha segment of 4 consecutive oxy-LNA-T monomers (Rajwanshi et al., Chem.Commun., 1999, 2073-74). Increased stability of oligonucleotidescontaining alpha-L-oxy-LNA monomers (^(Me)C, A, T-monomers) have beendemonstrated. The alpha-L-oxy-LNA monomers were incorporated intooligonucleotides with alternating alpha-L-oxy-LNA and DNA monomers(mix-mers) and in fully modified alpha-L-oxy-LNA oligomers. Thestability was compared to oxy-LNA and to DNA and it was found thatalpha-L-oxy-LNA monomers displaced the same protection pattern asoxy-LNA (Sørensen, et al., J. Amer. Chem. Soc., 2002, 124(10), 2164-76).The same alpha-L-oxy-LNA containing oligonucleotides were tested inRNase H assays and it was found that the designs disclosed were notefficiently recruiting RNase H. When these examples are taken togetheralso in combination with the data published by Arzumanov et al(Biochemistry 2001, 40, 14645-54) it has not been shown thatalpha-L-oxy-LNA containing oligonucleotides efficiently recruits RNaseH.

Oligonucleotides containing any combination of the diastereoisomers andany other LNA family member has not been demonstrated.

Natural dsDNA exists at physiological pH as a B-form helix, whereasdsRNA exists as an A-form helix. A helix formed by DNA and RNA exists inan intermediate A/B-form. This morphological difference is originated inthe difference in the preferred sugar conformations of the deoxyribosesand the riboses. The furanose ring of deoxyribose exists at roomtemperature in an equilibrium between C2′-endo (S-type) and C3′-endo(N-type) conformation with an energy barrier of ˜2 kcal/mol (FIG. 3).For deoxyribose the S-type conformation is slightly lowered in energy(˜0.6 kcal/mol) compared to the N-type and explains why DNA is found inthe S-type conformation. The conformation leads to the B-form helix. Forribose, and RNA, the preference is for the N-type that leads to theA-form helix. The A-form helix is associated with higher hybridisationstability. The oxy-LNA and the LNA analogues are locked in theN-conformation and consequently the oligonucleotides they are formingwill be RNA-like. The alpha-L-oxy-LNA is locked in a S-type andtherefore the oligonucleotides that it will form will be more DNA like(Sorensen et al., J. Amer. Chem. Soc., 2002, 124(10), 2164-76; Rajwanshiet al., Angew. Chem. Int. Ed., 2000; 39:1656-1659). Molecular strategiesare being developed to modulate unwanted gene expression that eitherdirectly causes, participates in, or aggravates a disease state. Onesuch strategy involves inhibiting gene expression with oligonucleotidescomplementary in sequence to the messenger RNA of a target gene. Themessenger RNA strand is a copy of the coding DNA strand and istherefore, as the DNA strand, called the sense strand. Oligonucleotidesthat hybridise to the sense strand are called antisenseoligonucleotides. Binding of these strands to mRNA interferes with thetranslation process and consequently with gene expression. Zamecnik andco-workers originally described the Antisense strategy and the principlehas since then attracted a lot of interest (Zamecnik & Stephenson, PNAS,1978, 75(1), 280-4; Bennet & Cowset, Biochim. Biophys. Acta, 1999, 1489,19-30; Crooke, 1998, Biotechnol. Genet. Eng Rev., 15, 121-57; Wengel, J.In Antisense Drug Technology; Principles, Strategies, and Applications;Edited by Crooke, S. T., Ed.; Marcel Dekker, Inc.: New York, Basel,2001; pp 339-357).

It has been a long sought goal to develop drugs with the capacity todestroy malignant genes base specifically. The applications of suchdrugs in e.g. cancer and infections diseases are self-evident. Nativeoligonucleotides cannot be employed as such mainly due to theirinstability in cellular media and to too low affinity for the targetgenes. The wish to develop nucleic acid probes with improved propertiesin this regard has been the main driver behind the massive synthesiseffort in the area of nucleic acid analogue preparation. The mostimportant guideline in this work has been to design the DNA analogues insuch a way that the DNA analogue would attain the N-type/“RNA”-likeconformation that is associated with the higher affinity of theoligonucleotides to nucleic acids.

One of the important mechanisms involved in Antisense is the RNase Hmechanism. RNase H is an intra cellular enzyme that cleaves the RNAstrand in RNA/DNA duplexes. Therefore, in the search for efficientAntisense oligonucleotides, it has been an important hallmark to prepareoligonucleotides that can activate RNase H. However, the prerequisitefor an oligonucleotide in this regard is therefore that the oligo isDNA-like and as stated above most high affinity DNA analogues inducesRNA-like oligonucleotides. Therefore, to compensate for the lack ofRNase H substrate ability of most DNA analogues (like e.g. 2′-OMe DNAanalogue and oxy-LNA) the oligonucleotides must havesegments/consecutive stretches of DNA and/or phosphorothioates.Depending on the design of the segments of such oligonucleotides theyare usually called Gap-mers, if the DNA segment is flanked by thesegments of the DNA analogue, Head-mers, if the segment of the DNAanalogue is located in the 5′ region of the oligonucleotide, andTail-mers, if the segment of the DNA analogue is located in the 3′region of the oligonucleotide.

It should be mentioned that other important mechanisms are involved inAntisense that are not dependent on RNase H activation. For sucholigonucleotides the DNA analogues, like LNA, can be placed in anycombination design (Childs et al. PNAS, 2002, 99(17), 11091-96; Crinelliet al., Nucl. Acid. Res., 2002, 30(11), 2435-43; Elayadi et al.,Biochemistry, 2002, 1, 9973-9981; Kurreck et al., Nucl. Acid. Res.,2002, 30(9), 1911-1918; Alayadi & Corey, Curr. opinion in Inves. Drugs.,2001, 2(4), 558-61; Braasch & Corey, Chem. & Biol., 2000, 55, 1-7).

In contrast to the beta-D-oxy-LNA the alpha-L-oxy-LNA has a DNA-likelocked conformation and it has been demonstrated that alpha-L-oxy-LNAcan activate RNase H (Sorensen et al., J. Amer. Chem. Soc., 2002,124(10), 2164-76). However, the cleavage rate of RNase H is much lowercompared to DNA in the disclosed designs and thus, the oligonucleotidesin the disclosed designs have not been shown to be efficient Antisensereagents.

SUMMARY OF THE INVENTION

The present inventors have found a novel class of pharmaceuticals whichcan be used in antisense therapy. Also, the inventors disclose noveloligonucleotides with improved antisense properties. The noveloligonucleotides are composed of at least one Locked Nucleic Acid (LNA)selected from beta-D-thio/amino-LNA or alpha-L-oxy/thio/amino-LNA. Theoligonucleotides comprising LNA may also include DNA and/or RNAnucleotides.

The present inventors have demonstrated that α-L-oxy-LNA surprisinglyprovides the possibility for the design of improved Antisenseoligonucleotides that are efficient substrates for RNase H. These noveldesigns are not previously described and the guidelines developedbroaden the design possibilities of potent Antisense oligonucleotides.Also comprised in this invention is the disclosure of Antisenseoligonucleotides having other improved properties than the capability ofbeing RNase H substrates. The oligonucleotides comprise any combinationof LNA-relatives with DNA/RNA, and their analogues, as well as oxy-LNA.The design of more potent Antisense reagents is a combination of severalfeatures. Among the features of these novel oligonucleotide designs areincreased enzymatic stability, increased cellular uptake, and efficientability to recrute RNase H. Also important is the relation between thelength and the potency of the oligonucleotides (e.g. a 15-mer having thesame potency as a 21-mer is regarded to be much more optimal). Thepotency of the novel oligonucleotides comprised in this invention istested in cellular in vitro assays and in vivo assays.It is furthermoreshowed that the novel designs also improves the in vivo properties suchas better pharmacokinetic/pharmacological properties and toxicityprofiles.

Beta-D-oxy-LNA and the analogues thio-and amino LNA:

LNA diastereoisomers:

Sugar conformations in DNA:

DESCRIPTION OF THE DRAWINGS

FIG. 1: Stability of oligonucleotides containing beta-D-amino-LNAagainst SVPD. (Capital letters are LNA, T^(N) stands forbeta-D-amino-LNA and small letters are DNA. The oligonucleotide issynthesized on deoxynucleoside-support.

FIG. 2A: Subcellular distribution in MiaPacaII cells of FAM-labeledoligonucleotide 2740 transfected with Lipofectamine2000

FIG. 2B: Subcellular distribution in MiaPacaII cells of FAM-labeledoligonucleotide 10 2774 transfected with Lipofectamine2000.

FIG. 2C: Subcellular distribution in MiaPacaII cells of FAM-labeledoligonucleotide 2752 transfected with Lipofectamine2000.

FIG. 2D: Subcellular distribution in MiaPacaII cells of FAM-labeledoligonucleotide 2746 transfected with Lipofectamine2000.

FIG. 3A: Uptake of titriated oligonucleotides (thio=2748; amino=2754;oxy=2742) in MiaPacaII cells at different oligonucleotide concentrationwith Lipofectamine2000 as 20 transfection agent.

FIG. 3B: Uptake of titriated oligonucleotides (thio=2748; amino=2754;oxy=2742) in 15PC3 cells at different oligonucleotide concentration withLipofectamine2000 as transfection agent.

FIG. 4: Down-regulation of Luciferase expression of oligonucleotidesgapmers containing beta-D-amino-LNA or beta-D-thio-LNA and thecorresponding beta-D-oxy-LNA gapmer control at 50 nM oligonucleotideconcentration.

FIG. 5A: Northern blot of oligonucleotides containing beta-D-amino-LNA(2754 and 2755), beta-D-thio-LNA (2748 and 2749) or beta-D-oxy-LNA(2742) at 400 and 800 nM in 15PC3 cells transfected withLipofectamine2000.

FIG. 5B: Quantification of Northern blot of oligonucleotides containingbeta-D-amino-LNA (2754 and 2755), beta-D-thio-LNA (2748 and 2749) orbeta-D-oxy-LNA (2742) at 400 and 800 nM in 15PC3 cells transfected withLipofectamine2000.

FIG. 5C: Northern blot of titration oligonucleotides containingbeta-D-amino-LNA (2754 and 2755), beta-D-thio-LNA (2748 and 2749) orbeta-D-oxy-LNA (2742).

FIG. 6: Northern blot analysis of oligonucleotides containingbeta-D-amino-LNA (2754), beta-D-thio-LNA (2748), alpha-L-oxy-LNA (2776)or beta-D-oxy-LNA (2742) at 50-400 nM in 15PC3 cells transfected withLipofectamine2000; comparison with the corresponding mismatch control at400 nM. Mismatch controls (thio=2750; amino=2756; alpha=2778) were alsoanalyzed at 30-90 nM and compared with the corresponding match at 30 nM.Table containing Northern blot analysis of oligonucleotides containingbeta-D-amino-LNA (2754), alpha-L-oxy-LNA (2776) and beta-D-oxy-LNA(2742) at 5-40 nM in 15PC3 cells transfected with Lipofectamine2000;comparison with the corresponding mismatch controls at 20 nM.

FIG. 7A: Serum clearance of titriated 2754=amino, 2748=thio and 2742=oxyafter 30 min of intravenous bolus injection. 2131 is an oligonucleotidegapmer containing beta-D-oxy-LNA used as a reference.

FIG. 7B: Biodistribution of titriated 2754=amino, 2748=thio and 2742=oxyafter 30 min of intravenous bolus injection. 2131 is an oligonucleotidegapmer containing beta-D-oxy-LNA used as a reference.

20

FIG. 7C: Specific tissue uptake of titriated 2754=amino, 2748=thio and2742=oxy after 30 min of intravenous bolus injection. 2131 is anoligonucleotide gapmer containing beta-D-oxy-LNA used as a reference.

FIG. 8A: Total uptake of titriated 2754=amino, 2748=thio and 2742=oxyafter 14 days of continuous administration at a 2.5 mg/Kg/day dosageusing Alzet osmotic minipumps.

FIG. 8B: Specific uptake of titriated 2754=amino, 2748=thio and 2742=oxyafter 14 days of continuous administration at a 2.5 mg/Kg/day dosageusing Alzet osmotic minipumps.

FIG. 9: Electrophoresis analysis of ³²P-labelled target RNA degradationproducts mediated by RNaseH and an oligonucleotide containingbeta-D-amino-LNA. Aliquots taken at 0, 10, 20 and 30 min for eachdesign. In the drawings, the line is DNA, the rectangle beta-D-amino- or-thio-LNA.

FIG. 10: Stability of oligonucleotides containing beta-D-thio-LNAagainst SVPD. (Capital letters are LNA, T^(S) stands for beta-D-thio-LNAand small letters are DNA. The oligonucleotide is synthesized ondeoxynucleoside-support, t.)

FIG. 11: FACS analysis of oligonucleotides containing beta-D-thio-LNAand the corresponding controls.

FIG. 12: Stability of oligonucleotides containing alpha-L-oxy-LNAagainst SVPD. (Capital letters are LNA, T^(α) stands for alpha-L-oxy-LNAand small letters are DNA. The oligonucleotide is synthesized ondeoxynucleoside-support, t.)

FIG. 13: Stability of different oligonucleotides (t₁₆, t_(s12), T₁₆,T^(α) ₁₅T) against S1-endonuclease. (Capital letters are LNA, T^(α)stands for alpha-L-oxy-LNA and small letters are DNA. Theoligonucleotide is synthesized on oxy-LNA-support, T.)

FIG. 14: FACS analysis of oligonucleotides containing alpha-L-oxy-LNA,and the corresponding controls.

FIG. 15: Gapmers including alpha-L-oxy-LNA (shadowed in gray).

FIG. 16: Down-regulation of Luciferase expression of oligonucleotidescontaining alpha-L-oxy-LNA at 50 nM oligonucleotide concentration.

FIG. 17A: Mixmers (4-3-1-3-5) containing alpha-L-oxy-LNA. The numbersstand for the alternate contiguous stretch of DNA or LNA. In thedrawing, the line is DNA, the rectangle beta-D-oxy-LNA, the gray shadowcorresponds to alpha-L-oxy-LNA residues.

FIG. 17B: Mixmers (4-1-1-5-1-1-3) containing alpha-L-oxy-LNA. Thenumbers stand for the alternate contiguous stretch of DNA or LNA. In thedrawing, the line is DNA, the rectangle beta-D-oxy-LNA, the gray shadowcorresponds to alpha-L-oxy-LNA residues.

FIG. 18A: Mixmers (4-1-5-1-5) containing alpha-L-oxy-LNA. The numbersstand for the alternate contiguous stretch of DNA or alpha-L-oxy-LNA. Inthe drawing, the line is DNA, the gray shadow corresponds toalpha-L-oxy-LNA residues.

FIG. 18B: Mixmers (3-3-3-3-1) containing alpha-L-oxy-LNA. The numbersstand for the alternate contiguous stretch of DNA or alpha-L-oxy-LNA. Inthe drawing, the line is DNA, the gray shadow corresponds toalpha-L-oxy-LNA residues.

FIG. 19A: Electrophoresis analysis of ³²P-labelled target RNAdegradation products mediated by RNaseH and an oligonucleotidecontaining alpha-L-oxy-LNA. Aliquots taken at 0, 10, 20 and 30 min foreach design. In the drawings, the line is DNA, the rectangebeta-D-oxy-LNA, the gray shadow corresponds to alpha-L-oxy-LNA residues.

FIG. 19B: Electrophoresis analysis of ³²P-labelled target RNAdegradation products mediated by RNaseH and an oligonucleotidecontaining alpha-L-oxy-LNA. Aliquots taken at 0, 10, 20 and 30 min foreach design. In the drawings, the line is DNA, the rectanglebeta-D-oxy-LNA, the gray shadow corresponds to alpha-L-oxy-LNA residues.

FIG. 20A: Tumor growth in nude mice treated with the indicated doses for14 days using Alzet osmotic minipumps for MiaPacaII cells.

FIG. 20B: Tumor growth in nude mice treated with the indicated doses for14 days using Alzet osmotic minipumps for MiaPacaII cells.

FIG. 20C: Tumor growth in nude mice treated with the indicated doses for14 days using Alzet osmotic minipumps for 15PC3 cells.

FIG. 20D: Tumor growth in nude mice treated with the indicated doses for14 days using Alzet osmotic minipumps for 15PC3 cells.

FIG. 21A: ASAT levels in mice serum after 14-day treatment using Alzetosmotic minipumps with the indicated oligonucleotides and at theindicated concentrations. 2722 and 2713 are oligonucleotides notrelevant to this study.

FIG. 21B: ALAT levels in mice serum after 14-day treatment using Alzetosmotic minipumps with the indicated oligonucleotides and at theindicated concentrations. 2722 and 2713 are oligonucleotides notrelevant to this study.

FIG. 21C: Alkaline phosphatase levels in mice serum after 14-daytreatment using Alzet osmotic minipumps with the indicatedoligonucleotides and at the indicated concentrations. 2722 and 2713 areoligonucleotides not relevant to this study.

FIG. 21D: Dosages used in studies in FIGS. 21A-21C.

FIG. 22: Monitoring the body temperature of the mice during the in vivoexperiment. 2722 and 2713 are oligonucleotides not relevant to thisstudy.

FIG. 23: Special constructs with beta-D-oxy-LNA. The numbers stand forthe alternate contiguous stretch of DNA and beta-D-oxy-LNA. In thedrawing, the line is DNA, the rectangle is beta-D-oxy-LNA.

FIG. 24: Down-regulation of Luciferase expression of special constructscontaining beta-D-oxy-LNA (designs 3-9-3-1) at 2 nM oligonucleotideconcentration.

DETAILED DESCRIPTION

Thus, the present invention in it broadest scope relates to apharmaceutical composition comprising a therapeutically active antisenseoligonucleotide construct which (i) comprises at least one lockednucleic acid unit selected from the group consisting of amino-LNA andthio-LNA and derivatives thereof; or (ii) comprises at least twoconsecutively located locked nucleotide units of which at least one isselected from the group consisting of alpha-L-oxy-LNA and derivativesthereof. The antisense construct can be in the form of a salt or in theform of prodrug or salts of such prodrug. The invention thus relates topharmaceutical compositions in which an active ingredient is apharmaceutically acceptable salt, prodrug (such as an ester) or salts ofsuch prodrug of the above oligonucleotide construct. Both amino- andthio-LNA can be either alpha or beta configuration, and in (i), theoligonucleotide construct encompasses constructs with at least one (suchas 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) units selected from the groupconsisting of: alpha-L-thio-LNA, beta-D-thio-LNA, beta-D-amino-LNA,alpha-L-amino-LNA and derivatives thereof; optionally in combinationwith at least one (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more) further independently selectedlocked or non-locked nucleotide units. Examples on these further unitsare oxy-LNA (such as alpha-L or beta-D), thio/amino LNA (such as alpha-Lor beta-D), a nucleotide unit which has a2′-deoxy-erythro-pentofuranosyl sugar moiety (such as a DNA nucleotide),a nucleotide unit which has a ribo-pentofuranosyl sugar moiety (such asa RNA nucleotide); and derivatives thereof. In (ii), the oligonucleotideconstruct encompasses constructs with at least two (such as 2, 3, 4, 5,6, 7, 8, 9, 10 or more) consecutively located nucleotide units, of whichat least one (such as 1, 2, 3, 4, 5, 6, 7 or more) is alpha-L-oxy LNAunits or derivatives thereof. In addition to the alpha-L-oxy LNA unitsor derivatives thereof, the sequence of consecutively located lockednucleotide units optionally comprises other locked nucleotide 35 units(such as the units defined herein). Besides the essential twoconsecutively located locked nucleotide units, the construct in (ii)optionally comprises one or more (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) independently selectedlocked or non-locked nucleotide units (such as the units definedherein).

In an interesting embodiment, the invention relates to a pharmaceuticalcomposition in which the antisense oligonucleotide construct comprisestwo adjacently located nucleotide sequences A and B, where

A represents a sequence of nucleotide units comprising (i) at least onelocked nucleotide unit selected from the group consisting of thio-LNA,amino-LNA (both in either alpha-L or beta-D configuration) andderivatives thereof, or (ii) at least two consecutively located lockednucleotide units of which at least one is selected from the groupconsisting of alpha-L-oxy-LNA and derivatives thereof; and

B represents one nucleotide unit or a sequence of nucleotide units, withthe proviso that at least one nucleotide unit in B has a2′-deoxy-erythro-pentofuranosyl sugar moiety or a ribo-pentofuranosylsugar moiety. Sequence A can additionally comprise at least one furtherlocked nucleotide unit (such as 2, 3, 4 or 5 units), preferably selectedindependently from the group consisting of amino-LNA, thio-LNA (both ineither alpha-L or beta-D configuration), alpha-L-oxy-LNA and derivativesthereof.

In an other interesting embodiment, the invention relates to apharmaceutical composition comprising an oligonucleotide construct whichcontains three adjacently located nucleotide sequences, A, B and C, inthe following order (5′ to 3′): A-B-C or C-B-A, in which

A represents a sequence comprising at least two consecutively locatedlocked nucleotide units, at least one of which is an alpha-L-oxy-LNAunit, and which sequence optionally contains one or more (such as 2, 3,4 or 5) non-locked nucleotide units (such as deoxyribonucleotide units,ribonucleotide units or derivatives thereof) and/or optionally containsone or more (such as 2, 3, 4 or 5) locked nucleotide units, such as aunit selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA(all in either alpha-L or beta-D configuration) and derivatives thereof;

B represents one nucleotide unit or a sequence of nucleotide units, withthe proviso that at least one nucleotide unit in B has a2′-deoxy-erythro-pentofuranosyl sugar moiety or a ribo-pentofuranosylmoiety; and

C represents a sequence comprising at least two consecutively locatedlocked nucleotide units, at least one of which is an alpha-L-oxy-LNAunit, and which sequence optionally contains one or more (such as 2, 3,4 or 5) non-locked nucleotide units (such as deoxyribonucleotide units,ribonucleotide units or derivatives thereof) and/or optionally containsone or more (such as 2, 3, 4 or 5) locked nucleotide units, such as aunit selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA(all in either alpha-L or beta-D configuration) and derivatives thereof.

The invention also relates to an oligonucleotide construct whichcomprises at least one nucleotide sequence comprising one or morenucleotide units selected from the group consisting of amino-LNA,thio-LNA (in all configurations) and derivatives thereof; with theproviso that the following oligonucleotide constructs are excluded:

(i) 5′-d(GTGAVATGC), 5′-d(GVGAVAVGC), 5′-d(GTGAXATGC), 5′-d(GXGAXAXGC),5′-d(GXGVXVXGC), in which sequences V represents a beta-D-amino-LNAthymine unit, and X represents a beta-D-methylamino-LNA thymine unit;and

(ii) 5′-d(GTGAYATGC), 5′-d(GYGAYAYGC) and 5′-d(GYGYYYYGC) in whichsequences Y represents a beta-D-thio-LNA uracil unit.

The excluded oligonucleotides are previously disclosed by Singh et aland Kumar et al. (Kumar et al. Bioorg, & Med. Chem. Lett.,1998, 8,2219-2222; Singh et al. J. Org. Chem., 1998, 63, 10035-39). It hascollectively for the excluded LNA-relatives been shown that they can beincorporated into oligonucleotides. However, no biological propertieshave not been demonstrated or suggested.

A presently preferred group of oligonucleotide constructs of theinvention comprises two adjacently located nucleotide sequences, A andB, where A represents a sequence of nucleotide units comprising at leastone locked nucleotide unit selected from the group consisting ofamino-LNA, thio-LNA (both in either alpha-L or beta-D) configuration,and derivatives thereof; and B represents one nucleotide unit or asequence of nucleotide units, with the proviso that at least onenucleotide unit in B has a 2′-deoxy-erythro-pentofuranosyl sugar moietyor a ribo-pentofuranosyl moiety; especially constructs in which Brepresents a sequence of nucleotide units, said sequence contains asubsequence of at least three nucleotide units having2′-deoxy-erythro-pentofuranosyl sugar moieties, such as 4, 5, 6, 7, 8, 9or 10 nucleotide units, said subsequence optionally being spiked with another nucleotide, preferably an alpha-L-oxy-LNA unit selected from thegroup consisting of alpha-L-amino-LNA, alpha-L-thio-LNA, alpha-L-oxy-LNAand derivatives thereof.

Also interesting is a construct according which comprises threeadjacently located nucleotide sequences in the following order (5′ to3′):A-B-C, in which the nucleotide sequences A and B are as defined asabove, and C represents a sequence of nucleotide units, which comprisesat least one locked nucleotide unit selected from the group consistingof amino-LNA, thio-LNA (both in either alpha-L or beta-D configuration)and derivatives thereof.

In the above constructs, it is preferred that A has a length of 2-10(preferably 2-8, such as 3, 4, 5, 6, 7) nucleotide units; B has a lengthof 1-10 (preferably 5-8, such as 6 or 7) nucleotide units; and C (ifpresent) has a length of 2-10 (preferably 2-8, such as 3, 4, 5, 6, or 7)nucleotide units; so that the overall length of the construct is 6-30(preferably 10-20, more preferably 12-18, such as 13, 14, 15, 16 or 17)nucleotide units.

A preferred embodiment of the above construct according to the inventionis a construct in which A represents a sequence of nucleotide unitscomprising at least two consecutively located locked nucleotide units(such as 3, 4, 5, 6, 7, 8, 9 or 10 units), at least one of said lockednucleotide units being selected from the group consisting of amino-LNA,thio-LNA and derivatives thereof; C represents a sequence of nucleotideunits comprising at least two consecutively located locked nucleotideunits (such as 3, 4, 5, 6, 7, 8, 9 or 10 units), at least one of saidlocked nucleotide units being selected from the group consisting ofamino-LNA, thio-LNA (in all configurations) and derivatives thereof,and/or B represents a sequence of least 2 nucleotide units (such as 3,4, 5, 6, 7, 8, 9 or 10 units), which sequence in addition to thenucleotide unit(s) having 2′-deoxy-erythro-pentofuranosyl sugarmoiety(ies) and/or ribo-pentofuranosyl moiety(ies), comprisesnucleotides units which are selected independently from the groupconsisting of: locked nucleotide units (such as alpha-L-oxy-, -thio-, or-amino-nucleotide units) and derivatives thereof.

An other embodiment of the invention relates to an oligonucleotideconstruct which contains three adjacently located nucleotide sequences,A, B and C, in the following order (5′ to 3′): A-B-C or C-B-A, in which

A represents a sequence comprising at least two consecutively locatedlocked nucleotide units, at least one of which is an alpha-L-oxy-LNAunit, and which sequence optionally contains one or more (such as 2, 3,4 or 5) non-locked nucleotide units (such as deoxyribonucleotide units,ribonucleotide units or derivatives thereof) and/or optionally containsone or more (such as 2, 3, 4 or 5) locked nucleotide units, such as aunit selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA(all in either alpha or beta configuration) and derivatives thereof;

B represents one nucleotide unit or a sequence of nucleotide units, withthe proviso that at 30 least one nucleotide unit in B has a2′-deoxy-erythro-pentofuranosyl sugar moiety or a ribo-pentofuranosylmoiety; and

C represents a sequence comprising at least two consecutively locatedlocked nucleotide units, at least one of which is an alpha-L-oxy-LNAunit, and which sequence optionally contains one or more (such as 2, 3,4 or 5) non-locked nucleotide units (such as deoxyribonucleotide units,ribonucleotide units or derivatives thereof) and/or optionally containsone or more (such as 2, 3, 4 or 5) locked nucleotide units, such as aunit selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA(all in either alpha or beta configuration) and derivatives thereof. Itis preferred that A has a length of 2-10 (preferably 2, 3, 4, 5, 6, 7,or 8) nucleotide units; B has a length of 1-10 (preferably 5, 6, 7, or8) nucleotide units;

C has a length of 2-10 (preferably 2, 3, 4, 5, 6, 7, or 8) nucleotideunits; so that the overall length of the construct is 8-30 (preferably10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) nucleotide units.

An other interesting embodiment is a construct in which A represents asequence of nucleotide units comprising at least three consecutivelylocated locked nucleotide units, at least one of said locked nucleotideunits being selected from the group consisting of alpha-L-oxy-LNA andderivatives thereof; C represents a sequence of nucleotide unitscomprising at least three consecutively located locked nucleotide units,at least one of said locked nucleotide units being selected from thegroup consisting of alpha-L-oxy-LNA and derivatives thereof; and/or Brepresents a sequence of least 2 nucleotide units (such as 3, 4, 5, 6,7, 8, 9 or 10 units), which sequence in addition to the nucleotideunit(s) having 2′-deoxy-erythro-pentofuranosyl sugar moiety(ies) and/orribo-pentofuranosyl moiety(ies), comprises nucleotide units which areselected independently from the group consisting of: locked nucleotideunits (such as alpha-L-oxy-, -thio-, or -amino-nucleotide units) andderivatives thereof. Especially preferred is a construct in which A andC comprises at least one alpha-L-oxy-LNA or alpha-L-thio-LNA unitlocated adjacent to B.

20

In a further embodiment, the invention relates to an oligonucleotidewhich has the formula (in 5′ to 3′ order): A-B-C-D, in which Arepresents a sequence of locked nucleotide units; B represents asequence of non-locked nucleotide units, preferably at least one unithas a 2′-deoxy pentofuranose sugar moiety, in which sequence 1 or 2nucleotide units optionally are substituted with locked nucleotideunits, preferably alpha-L-oxy-LNA; C represents a sequence of lockednucleotide units; and D represents a non-locked nucleotide unit or asequence of non-locked nucleotide units. It is preferred that A has alength of 2-6 (preferably 3, 4 or 5) nucleotide units; B has a length of4-12 (preferably 6, 7, 8, 9, 10 or 11) nucleotide units; C has a lengthof 1-5 (preferably 2, 3, or 4) nucleotide units; D has a lenght of 1-3(preferably 1-2) nucleotide units; and that the overall length of theconstruct is 8-26 (preferably 12-21) nucleotide units. In presentlypreferred construct, A has a length of 4 nucleotide units; B has alength of 7-9, preferably 8, nucleotide units; C has a length of 3nucleotide units; D has a length of 1 nucleotide unit; and the overalllength of the construct is 15-17 (preferably 16) nucleotide units. It isfurther preferred that the locked nucleotide units in A and C arebeta-D-oxy-LNA units or derivatives thereof.

The oligonucleotide constructs according to the invention can containnaturally occurring phosphordiester internucleoside linkages, as well asother internucleoside linkages as defined in this specification.Examples on internucleoside linkages are linkages selected from thegroup consisting of —O—P(O)₂—O—, —O—P(O,S)—O—, —O—P(S)₂—O—,—NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))—O—, —O—PO(R″)—O—, —O—PO(CH₃)—O—, and—O—PO(NHR^(N))—O—, where R^(H) is selected form hydrogen and C₁₋₄-alkyl,and R″ is selected from C₁₋₆-alkyl and phenyl.

In a further embodiment, the invention relates to an oligonucleotideconstruct which comprises at least one locked nucleotide unit selectedfrom the group consisting of amino-LNA, thio-LNA (both in either alpha-Lor beta-D configuration), alpha-L-oxy-LNA, and derivatives thereof;wherein at least one of the linkages between the nucleotide units isdifferent from the natural occurring phosphordiester (—O—P(O)₂—O—)linker. Constructs in which the internucleoside linkage (between 3′carbon and 5′ carbon on adjacent (3′, 5′ dideoxy) nucleosides) selectedfrom the group consisting of: —O—P(O,S)—O—, —O—P(S)2—O—,—NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))—O—, —O—PO(R″)—O—, —O—PO(CH₃)—O—, and—O—PO(NHR^(N))—O—, where R^(H) is selected form hydrogen and C₁₋₄-alkyl,and R″ is selected from C₁₋₆-alkyl and phenyl, is presently preferred,and the phoshorothioate internucleoside linkage is presently mostpreferred.

An embodiment of the oligonucleotide constructs according to theinvention relates to such constructs that are able to mediate enzymaticinactivation (at least partly) of the target nucleic acid (eg. a RNAmolecule) for the construct. Constructs that mediate RNase H cutting ofthe target are within the scope of the present invention. Thus, thepresent invention relates to constructs that are able to recruit RNase,especially constructs in which sequence B represents a sequence ofnucleotide units that makes the construct able to recruit RNase H whenhybridised to a target nucleic acid (such as RNA, mRNA).

It should be understood that the invention also relates to apharmaceutical composition which comprises a least one antisenseoligonucleotide construct of the invention as an active ingredient. Itshould be understood that the pharmaceutical composition according tothe invention optionally comprises a pharmaceutical carrier, and thatthe pharmaceutical composition optionally comprises further antisensecompounds, chemotherapeutic compounds, antiinflammatory compounds and/orantiviral compounds.

The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be (a) oral (b) pulmonary, e.g., by inhalation orinsufflation of powders or aerosols, including by nebulizer;intratracheal, intranasal, (c) topical including epidermal, transdermal,ophthalmic and to mucous membranes including vaginal and rectaldelivery; or (d) parenteral including intravenous, intraarterial,subcutaneous, intraperitoneal or intramuscular injection or infusion; orintracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, sprays, suppositories, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Preferred topical formulations include those inwhich the oligonucleotides of the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Compositionsand formulations for oral administration include but is not restrictedto powders or granules, microparticulates, nanoparticulates, suspensionsor solutions in water or non-aqueous media, capsules, gel capsules,sachets, tablets or minitablets. Compositions and formulations forparenteral, intrathecal or intraventricular administration may includesterile aqueous solutions which may also contain buffers, diluents andother suitable additives such as, but not limited to, penetrationenhancers, carrier compounds and other pharmaceutically acceptablecarriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels and suppositories. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances which increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

The antisense nucleotide constructs of the invention encompass, in theirbrodest scope, any pharmaceutically acceptable salts, esters, or saltsof such esters. Furthermore encompasses the invention any othercompound, which, upon administration to an animal or a human, is capableof directly or indirectly providing the biologically active metaboliteor residue thereof. The invention therefore also encompasses prodrugs ofthe compounds of the invention and pharmaceutically acceptable salts ofsuch prodrugs, and other bioequivalents. The term prodrug indicates atherapeutic agent that is prepared in an inactive form and that isconverted to an active form, a drug, within the body or cells thereof.The pharmaceutically acceptable salts include but are not limited tosalts formed with cations; acid addition salts formed with inorganicacids salts formed with organic acids such as, and salts formed fromelemental anions.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides, to the skin of animals or humans. Mostdrugs are present in solution in both ionized and nonionized forms.However, usually only lipid soluble or lipophilic drugs readily crosscell membranes. It has been discovered that even non-lipophilic drugsmay cross cell membranes if the membrane to be crossed is treated with apenetration enhancer. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs.

Pharmaceutical compositions of the invention include a pharmaceuticalcarrier that may contain a variety of components that provide a varietyof functions, including regulation of drug concentration, regulation ofsolubility, chemical stabilization, regulation of viscosity, absorptionenhancement, regulation of pH, and the like. The pharmaceutical carriermay comprise a suitable liquid vehicle or excipient and an optionalauxiliary additive or additives. The liquid vehicles and excipients areconventional and commercially available. Illustrative thereof aredistilled water, physiological saline, aqueous solutions of dextrose,and the like. For water soluble formulations, the pharmaceuticalcomposition preferably includes a buffer such as a phosphate buffer, orother organic acid salt. For formulations containing weakly solubleantisense compounds, micro-emulsions may be employed. Other componentsmay include antioxidants, such as ascorbic acid, hydrophilic polymers,such as, monosaccharides, disaccharides, and other carbohydratesincluding cellulose or its derivatives, dextrins, chelating agents, andlike components well known to those in the pharmaceutical sciences. Theoligonucleotides may be encapsulated in liposomes for therapeuticdelivery.

In a certain embodiment, the present invention provides pharmaceuticalcompositions containing (a) one or more antisense compounds and (b) oneor more other chemotherapeutic agents which function by a non-antisensemechanism. When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., mithramycin andoligonucleotide), sequentially (e.g., mithramycin and oligonucleotidefor a period of time followed by another agent and oligonucleotide), orin combination with one or more other such chemotherapeutic agents or incombination with radiotherapy.

Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs, mayalso be combined in compositions of the invention. Two or more combinedcompounds may be used together or sequentially.

In another embodiment, compositions of the invention may contain one ormore antisense compounds, particularly oligonucleotides, targeted to afirst nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Two or more combined compoundsmay be used together or sequentially.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, and the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.

Optimum dosages may vary depending on the relative potency of individualoligonucleotides. Generally it can be estimated based on EC50s found tobe effective in in vitro and in vivo animal models. In general, dosageis from 0.01 ug to 25 g per kg of body weight, and may be given once ormore daily, weekly, monthly or yearly, or even once every 2 to 10 years.The repetition rates for dosing can be estimated based on measuredresidence times and concentrations of the drug in bodily fluids ortissues. Following successful treatment, it may be desirable to have thepatient undergo maintenance therapy to prevent the recurrence of thedisease state.

The LNA containing antisense compounds of the present invention can beutilized for diagnostics, therapeutics, prophylaxis and as researchreagents and kits. For therapeutics, an animal or a human, suspected ofhaving a disease or disorder, which can be treated by modulating theexpression of a gene by administering antisense compounds in accordancewith this invention. Further provided are methods of treating an animaland humans, suspected of having or being prone to a disease orcondition, associated with expression of a target gene by administeringa therapeutically or prophylactically effective amount of one or more ofthe antisense compounds or compositions of the invention. Examples ofsuch a diseases are for example different types of cancer, infectiousand inflammatory diseases.

In a certain embodiment, the present invention relates to a method ofsynthesis of a pharmaceutical compositions, a oligonucleotides or aconstruct according to the present invention.

Definitions

The term “nucleotide sequence” or “sequence” comprises a plurality (ie.more than one) nucleosides (or derivatives thereof), in which sequenceeach two adjacent nucleosides (or derivatives thereof) are linked by aninternucleoside linker. When the length of a sequence are defined by arange (such as from 2-10 nucleotide units), the range are understood tocomprise all integers in that range, i.e. “a sequence of 2-10 nucleotideunits” comprises sequences having 2, 3, 4, 5, 6, 7, 8, 9, or 10nucleotide units.

In the present context, the term “oligonucleotide” (or oligo, oligomer)means a successive chain of nucleoside units (i.e. glycosides ofheterocyclic bases) connected via internucleoside linkages.

By the term “unit” is understood a monomer.

The term “at least one” comprises the integers larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 andso forth.

The term “locked nucleotide” comprises nucleotides in which the 2′ deoxyribose sugar moiety is modified by introduction of a structurecontaining a heteroatom bridging from the 2′ to the 4′ carbon atoms. Theterm includes nucleotides having the following substructures (the oxygenat the 3′ and 5′ ends illustrates examples of the starting point of theinternucleoside linkages):

beta-D-LNA derivatives:

alpha-L-LNA derivatives

In both structures, X represents O, S or N—R (R═H; C1-C6 alkyl such asmethyl, ethyl, propyl, i-propyl, butyl, i-butyl, t-butyl and pentyl);and

n is an integer 1, 2 or 3, so that the group —(CH2)n- comprisesmethylen, ethylen or propylen groups. In these alkylene groups (and the—N(C1-C6 alkyl)- group), one or more H atoms can be replaced withsubstituents, such as one or more substituents selected from the groupconsisting of halogen atoms (Cl, F, Br, I), Nitro, C1-6 alkyl or C1-6alkoxy, both optionally halogenated.

10 In the present context, the term “C₁₋₆-alkyl” means a linear, cyclicor branched hydrocarbon group having 1 to 6 carbon atoms, such asmethyl, ethyl, propyl, iso-propyl, butyl, tert-butyl, iso-butyl, pentyl,cyclopentyl, hexyl, cyclohexyl, in particular methyl, ethyl, propyl,iso-propyl, tert-butyl, iso-butyl and cyclohexyl. “C₁₋₆-alkoxy” means—O—(C1-6-alkyl).

The term “non-locked nucleotide” comprises nucleotides that do notcontain a bridging structure in the ribose sugar moiety. Thus, the termcomprises DNA and RNA nucleotide monomers (phosphorylated adenosine,guanosine, uridine, cytidine, deoxyadenosine, deoxyguanosine,deoxythymidine, deoxycytidine) and derivatives thereof as well as othernucleotides having a 2′-deoxy-ervthro-pentofuranosyl sugar moiety or aribo-pentofuranosyl moiety.

The term “thio-LNA” comprises a locked nucleotide in which X in theabove formulas represents S, and n is 1. Thio-LNA can be in both beta-Dand alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which X in theabove formulas represents —NR—, and n is 1. Amino-LNA can be in bothbeta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which X in the aboveformulas represents O and n is 1. Oxy-LNA can be in both beta-D andalpha-L-configuration.

By the term “alpha-L-LNA” as used herein is normally understoodalpha-L-oxy-LNA (n=1 in the bridging group), and by the term “LNA” asused herein is understood beta-D-oxy-LNA monomer wherein n in thebridging group is 1.

However, derivatives of the above locked LNA's comprise nucleotides inwhich n is an other integer than 1.

By the term “derivatives thereof” in connection with nucleotides (e.g.LNA and derivatives thereof) is understood that the nucleotide, inaddition to the bridging of the furan ring, can be further derivatized.For example, the base of the nucleotide, in addition to adenine,guanine, cytosine, uracil and thymine, can be a derivative thereof, orthe base can be substituted with other bases. Such bases includesheterocyclic analogues and tautomers thereof. Illustrative examples ofnucleobases are xanthine, diaminopurine, 8-oxo-N⁶-methyladenine,7-deazaxanthine, 7-deazaguanine, N⁴,N⁴-ethanocytosin,N⁶,N⁶-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C³-C⁶)-alkynylcytosine, 5-fluorouracil, 5-bromouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridin, isocytosine,isoguanin, inosine, N⁶-alylpurines, N⁶-acylpurines, N⁶-benzylpurine,N⁶-halopurine, N⁶-vinylpurine, N⁶-acetylenic purine, N⁶-acyl purine,N⁶-hydroxyalkyl purine, N⁶-thioalkyl purine, N²-alkylpurines,N⁴-alkylpyrimidines, N⁴-acylpyrimidines, N⁴-benzylpurine,N⁴-halopyrimidines, N⁴-vinylpyrimidines, N⁴-acetylenic pyrimidines,N⁴-acyl pyrimidines, N⁴-hydroxyalkyl pyrimidines, N⁶-thioalkylpyrimidines, thymine, cytosine, 6-azapyrimidine, including6-azacytosine, 2- and/or 4-mercaptopyrimidine, uracil,C⁵-alkylpyrimidines, C⁵-benzylpyrimidines, C⁵-halopyrimidines,C⁵-vinylpyrimidine, C⁵-acetylenic pyrimidine, C⁵-acyl pyrimidine,C⁵-hydroxyalkyl purine, C⁵-amidopyrimidine, C⁵-cyanopyrimidine,C⁵-nitropyrimidine, C⁵-aminopyrimdine, N²-alkylpurines, N²-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl, trazolopyridinyl,imidazolopyridinyl, pyrrolopyrimidinyl, and pyrazolopyrimidinyl.Functional oxygen and nitrogen groups on the base can be protected asnecessary or desired. Suitable protecting groups are well known to thoseskilled in the art, and included trimethylsilyl, dimethylhexylsilyl,t-butyldimenthylsilyl, and t-butyldiphenylsilyl, trityl, alkyl groups,acyl groups such as acetyl and propionyl, methanesulfonyl, andp-toluenesulfonyl. Preferred bases include cytosine, methyl cytosine,uracil, thymine, adenine and guanine. In addition to the derivatisationof the base, both locked and non-locked nucleotides can be derivatisedon the ribose moiety. For example, a 2′ substituent can be introduced,such as a substituent selected from the group consisting of halogen(such as fluor), C1-C9 alkoxy (such as methoxy, ethoxy, n-propoxy ori-propoxy), C1-C9 aminoalkoxy (such as aminomethoxy and aminoethoxy),allyloxy, imidazolealkoxy, and polyethyleneglycol, or a 5′ substituent(such as a substituent as defined above for the 2′ position) can beintroduced.

By the terms “internucleoside linkage” and “linkage between thenucleotide units” (which is used interchangeably) are to be understoodthe divalent linker group that forms the covalent linking of twoadjacent nucleosides, between the 3′ carbon atom on the first nucleosideand the 5′ carbon atom on the second nucleoside (said nucleosides being3′, 5′ dideoxy). The oligonucleotides of the present invention comprisessequences in which both locked and non-locked nucleotides independentlycan be derivatised on the internucleoside linkage which is a linkageconsisting of preferably 2 to 4 groups/atoms selected from —CH₂—, —O—,—S—, —NR^(H)—, >C═O, >C═NR^(H), >C═S, —Si(R″)₂—, —SO—, —S(O)₂—, —P(O)₂—,—PO(BH₃)—, —P(O,S)—, —P(S)₂—, —PO(R″)—, —PO(OCH₃)—, and —PO(NHR^(H))—,where R^(H) is selected form hydrogen and C₁₋₆-alkyl, and R″ is selectedfrom C₁₋₆-alkyl and phenyl. Illustrative examples of suchinternucleoside linkages are —CH₂—CH₂—CH₂—, —CH₂—CO—CH₂—,—CH₂—CHOH—CH₂—, —O—CH₂—, —O—CH₂—CH₂—, —O—CH₂—CH(R5)-, —CH₂—CH₂—O—,—NR^(H)—CH₂—CH₂—, —CH₂—CH₂—NR^(H)—, —CH₂—NR^(H)—CH₂—,—O—CH₂—CH₂—NR^(H)—, —NR^(H)—CO—O—, —NR^(H)—CO—NR^(H)—,—NR^(H)—CS—NR^(H)—, —NR^(H)—C(═NR^(H))—NR^(H)—, —NR^(H)—CO—CH₂—NR^(H)—,—O—CO, —O—CO—CH₂—O—, —O—CH₂—CO—O—, —CH₂—CO—NR^(H)—, —O—CO—NR^(H)—,—NR^(H)—CO—CH₂—, —O—CH₂—CO—NR^(H)—, —O—CH₂—CH₂—NR^(H)—, —CH═N—O—,—CH₂—NR^(H)—O—, —CH₂—O—N(R5)-, —CH₂—O—NR^(H)—, —CO—NR^(H)—CH₂—,—CH₂—NR^(H)—O—, —CH₂—NR^(H)—CO—, —O—NR^(H)—CH₂—, —O—NR^(H)—, —O—CH₂—S—,—S—CH₂—O—, —CH₂—CH₂—S—, —O—CH₂—CH₂—S—, —S—CH₂—CH(R5)-, —S—CH₂—CH₂—,—S—CH₂—CH₂—O—, —S—CH₂—CH₂—S—, —CH₂—S—CH₂—, —CH₂—SO—CH₂—, —CH₂—SO₂—CH₂—,—O—SO—O—, —O—S(O)₂—O—, —O—S(O)₂—CH₂—, —O—S(O)₂—NR^(H)—,—NR^(H)—S(O)₂—CH₂—, —O—S(O)₂—CH₂—, —O—P(O)₂—O—, —O—P(O,S)—O—,—O—P(S)₂—O—, —S—P(O)₂—O—, —S—P(O,S)—O—, —S—P(S)₂—O—, —O—P(O)₂—S—,—O—P(O,S)—S—, —O—P(S)₂—S—, —S—P(O)₂—S—, —S—P(O,S)—S—, —S—P(S)₂—S—,—O—PO(R″)—O—, —O—PO(OCH₃)—O—, —O—PO(OCH₂CH₃)—O—, —O—PO(OCH₂CH₂S—R)—O—,—O—PO(BH₃)—O—, —O—PO(NHR^(N))—O—, —O—P(O)₂—NR^(H)—, —NR^(H)—P(O)₂—O—,—O—P(O,NR^(H))—O—, —CH₂—P(O)₂—O—, —O—P(O)₂—CH₂—, and —O—Si(R″)₂—O—;where R5 is selected from hydrogen and C₁₋₆-alkyl, R^(H) is selectedform hydrogen and C₁₋₆-alkyl, and R″ is selected from C₁₋₆-alkyl andphenyl.

—CH₂—CO—NR^(H)—, —CH₂—NR^(H)—O—, —S—CH₂—O—, —O—P(O)₂—O—, —O—P(O,S)—O—,—O—P(S)₂—O—, —NR^(H)—P(O)₂—O—, —O—P(O,NR^(H))—O—, —O—PO(R″)—O—,—O—PO(CH₃)—O—, and —O—PO(NHR^(N))—O—, where R^(H) is selected fromhydrogen and C₁₋₆-alkyl, and R″ is selected from C₁₋₆-alkyl and phenyl,are especially preferred.

The nucleotides units may also contain a 3′-Terminal group or a5′-terminal group, preferably —OH.

By the term “able to recruit RNase H” is understood that the anoligonucleotide construct, in order to elicit RNase H enzyme cleavage ofa target nucleic acid (such as target mRNA), 35 must include a segmentor subsequence that is of DNA type. This means that at least somenucleotide units of the oligonucleotide construct (or a subsequencethereof) must have 2′-deoxy-erythro-pentofuranosyl sugar moieties. Asubsequence having more than three consecutive, linked2′-deoxy-erythro-pentofuranosyl containing nucleotide units likely isnecessary in order to elicit RNase H activity upon hybridisation of anoligonucleotide construct of the invention with a target nucleic acid,such as a RNA. Preferably, a sequence which is able to recruit RNase Hcontains more than three consecutively located nucleotides having2′-deoxy-erythro-pentofuranosyl sugar moieties, such as 4, 5, 6, 7, 8 ormore units. However, such a subsequence of consecutively locatednucleotides having 2′-deoxy-erythro-pentofuranosyl sugar moieties can byspiked (ie. one or more (such as 1, 2, 3, 4, or more) nucleotides beingreplaced) with other nucleotides, preferably alpha-L-oxy, thio- oramino-LNA units or derivatives thereof.

The term “pharmaceutically acceptable salt” is well known to the personskilled in the art.

Examples of such pharmaceutically acceptable salts are the iodide,acetate, phenylacetate, trifluoroacetate, acrylate, ascorbate, benzoate,chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate,methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, bromide,isobutyrate, phenylbutyrate, g-hydroxybutyrate, b-hydroxybutyrate,butyne-1,4-dioate, hexyne-1,4-dioate, hexyne-1,6-dioate, caproate,caprylate, chloride, cinnamate, citrate, decanoate, formate, fumarate,glycollate, heptanoate, hippurate, lactate, malate, maleate,hydroxymaleate, malonate, mandelate, mesylate, nicotinate,isonicotinate, nitrate, oxalate, phthalate, terephthalate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, propiolate, propionate, phenylpropionate, salicylate,sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite,bisulfite, sulfonate, benzenesulfonate, p-bromophenylsulfonate,chlorobenzenesulfonate, propanesulfonate, ethanesulfonate,2-hydroxyethanesulfonate, methanesulfonate, naphthalene-l-sulfonate,naphthalene-2-sulfonate, p-toluenesulfonate, xylenesulfonate, tartarate,and the like.

Experimental

Oligonucleotide Synthesis

Oligonucleotides were synthesized using the phosphoramidite approach onan Expedite 8900/MOSS synthesizer (Multiple Olionucleotide SynthesisSystem) at 1 μM scale. At the end of the synthesis (DMT-on) theoligonucleotides were cleaved from the solid support using aqueousammonia for 1 h at room temperature, and further deprotected for 4 h at65° C. The crudes were purified by reverse phase HPLC. After the removalof the DMT-group, the oligonucleotides were characterized by AE-HPLC orRP-HPLC, and the structure further confirmed by ESI.

3′-Exonuclease Stability Study

Snake venom phosphodiesterase (SVPD, Amersham Pharmacia) assays wereperformed using 26 μg/mL oligonucleotide, 0.3 μg/mL enzyme at 37° C. ina buffer of 50 mM Tris-HCl, 10 mM MgCl₂, pH 8. The enzyme was shown tomaintain its activity under these conditions for at least 2 h. Aliquotsof the enzymatic digestion were removed at the indicated times, quenchedby heat denaturation for 3 min and stored at −20° C. until analysis byRP-HPLC.

S1—Endonuclease Stability Study

S1 endonuclease (Amersham Pharmacia) assays were performed using 1.5μmol oligonucleotide and 16 U/mL enzyme at 37° C. in a buffer of 30 mMNaOAc, 100 mM NaCl, 1 mM ZnSO₄, pH 4.6. The enzyme was shown to maintainits activity under these conditions for at least 2 h. Aliquots of theenzymatic digestion were removed at the indicated times, quenched byfreezing-drying, and stored at −20° C. until analysis by either RP-HPLCand ES-MS or polyacrylamide electrophoresis.

Luciferase Assay

The X1/5 Hela cell line (ECACC Ref. No: 95051229), which is stablytransfected with a “tet-off” luciferase system, was used. In the absenceof tetracycline the luciferase gene is expressed constitutively. Theexpression can be measured as light in a luminometer, when theluciferase substrate, luciferin has been added.

The X1/5 Hela cell line was grown in Minimun Essential Medium Eagle(Sigma M2279) supplemented with 1× Non Essential Amino Acid (SigmaM7145), 1× Glutamax I (Invitrogen 35050-038), 10% FBS calf serum, 25μg/ml Gentamicin (Sigma G1397), 500 μg/ml G418 (Invitrogen 10131-027)and 300 μg/ml Hygromycin B (invitrogen 10687-010). The X1/5 Hela cellswere seeded at a density of 8000 cells per well in a white 96 well plate(Nunc 136101) the day before the transfection. Before the transfection,the cells were washed one time with OptiMEM (Invitrogen) followed byaddition of 40 μl of OptiMEM with 2 μg/ml of Lipofectamine2000(Invitrogen). The cells were incubated for 7 minutes before addition ofthe oligonucleotides. 10 μl of oligonucleotide solutions were added andthe cells were incubated for 4 hours at 37° C. and 5% CO2. After the 4hours of incubation the cells were washed once in OptiMEM and growthmedium was added (100 μl). The luciferase expression was measure thenext day.

Luciferase expression was measured with the Steady-Glo luciferase assaysystem from Promega. 100 μl of the Steady-Glo reagent was added to eachwell and the plate was shaken for 30 s at 700 rpm. The plate was read inLuminoskan Ascent instrument from ThermoLabsystems after 8 min ofincubation to complete total lysis of the cells. The luciferaseexpression is measured as Relative Light Units per seconds (RLU/s). Thedata was processed in the Ascent software (v2.6) and graphs were drawnin SigmaPlot2001.

RNaseH assay

25 nM RNA was incubated in the presence of a 10-fold excess of variouscomplementary oligonucleotides in 1× TMK-glutamate buffer (20 mM Trisacetate, 10 mM magnesium acetate and 200 mM potassium glutamate, pH7.25) supplied with 1 mM DTT in a reaction volume of 40 μl. Thereactions were preincubated for 3 minutes at 65° C. followed by 15minutes at 37° C. before addition of RNase H (Promega, Cat.#4285). 0.2 Uof RNase H was added, and samples were withdrawn (6 μl) to formamide dye(3 μl) on ice at the time points 0, 10, 20 and 30 minutes after RNase Haddition. 3 μl of the 0, 10, 20 and 30 minutes samples were loaded on a15% polyacrylamide gel containing 6M urea and 0.9× Tris borate/EDTAbuffer. The gel was 0.4 mm thick and ran at 35 watt as the limitingparameter for 2 hours. The gel was dried for 60 minutes at 80° C.,followed by ON exposure on Kodak phosphorscreen. The Kodakphosphorscreen was read in a Bio-Rad FX instrument and the result wasanalysed in Bio-Rad software Quantity One.

Cellular Assay: Luciferase Target

Cell Culture: Cell lines 15PC3 (human prostate cancer) and X1/5 (HeLacells stably transfected with a Tet-Off luciferase construct) were used,15PC3 were kindly donated by F. Baas, Neurozintuigen lab, Amsterdam, TheNetherlands, X1/5 were purchased from ECACC. 15PC3 were maintained inDMEM+10% FCS+glutamax+gentamicin and X1/5 were maintained in DMEM+10%FCS+glutamax+gentamicin+hygromycin+G418 and both cell lines werepassaged twice weekly.

Transfection: Cells were seeded at 150000 cells pr. well in 12-wellplates the day before transfection. For transfection with lipid,Lipofectamine 2000 (GIBCO BRL) was mixed with OptiMem and 300 μl of themixture was added to each well and incubated for 7 min. before additionof 100 μl oligo diluted in OptiMem. For each cell line, the optimalLipofectamine 2000 was determined, for X1/5, the optimal Lipofectamineconcentration was 2 μg/ml and for 15PC3 the optimal concentration was 10μg/ml.

For transfection without lipid, the cells were washed in OptiMem (GIBCOBRL) and 300 μl OptiMem was added to each well. Working stocks of 200 μMwere prepared of each oligonucleotide to be tested and added to eachwell obtaining the desired concentration. For mock controls,oligonucleotide was substituted with water in both protocols. The cellswere incubated with the oligonucleotide for 4 h at 37° C. and 5% CO2 ina humidified atmosphere and subsequently washed in OptiMem beforecomplete growth medium was added. The cells were incubated for anadditional 20 h.

For FACS analysis, cells were harvested by trypsination and washed twicein Cell Wash (BD) and resuspended in lx Cell Fix (BD).

FACS analysis: FACS analysis was performed on a FACSCalibur (BD),settings were adjusted on mock controls. Data analysis was performedusing the Cell Quest Pro software (BD).

Assisted Cellular Uptake

Transfections were performed in 6 well culture plates on microscopeglass coverslips with FAM-labeled oligonucleotides at 400 nM.Transfections were done with either DAC30 (Eurogentec) or Lipofectamine2000 as liposomal transfection agents for 5 h in serum free DMEM at 37°C. Immediately after the transfection period, the cells were washed withPBS and fixed with 4% paraformaldehyde.

Cell Lines: Ha-Ras Target

Prostate cancer cell line 15PC3 and pancreatic carcinoma cell lineMiaPacaII were maintained by serial passage in Dulbecco's modifiedEagle's medium (DMEM). Cells were grown at 37° C. and 5% CO2. Media weresupplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/mLpenicillin and 100 μg/mL streptomycin.

Transfections: Ha-Ras Target

Cell transfections were performed with 15PC3 cells plated in 6 wellculture plates. The cells were plated (70% confluent) the day beforetransfection. The transfections were usually performed usinglipofectamine 2000 (Invitrogen) according to the manufacturer'sinstructions, except for using serum free DMEM. Cells were transfectedfor 5 hours. Afterwards the medium was replaced with fresh DMEM. We alsocompared Lipofectamine 2000 with DAC30 (Eurogentec). When DAC30 was usedthe protocol described in Ten Asbroek et al. (NAR 28, 1133-1138) wasfollowed.

For Fluorescence studies the cells were plated on glass cover slips in 6well culture plates. Transfections were performed as described above butusing FAM labeled oligonucleotides. At the time of analysis, the cellswere fixed on the glass in 4% paraformaldehyde and sealed on microscopeglass in Vectashield mounting medium (Vector Laboratories Inc.).

Fluorescence microscopy was done with a Vanox Microscope and appropriatefiltres.

mRNA Analysis: Ha-Ras Target

After 20 hours the cells were harvested in TRIZOL (Invitrogen), 1 ml perwell.

The RNA was isolated according to the manufacturer's instructions forTRIZOL. The RNA was separated on glyoxal gels containing 1% agarosefollowing standard protocols. RNA was subsequently blotted onto HybondN+ membrane (Amersham) in 20×SSC. After the transfer, the RNA was UVcross-linked, and then the membrane was baked for 2 hours at 80° C.Hybridizations and post-hybridization washes were done according toChurch and Gilbert (PNAS 81, 1991-1995). The Ha-Ras probe used wasgenerated using Ha-Ras primers according to Sharpe et al. (J. AM. Soc.Nephrol. 11 1600-1606) cloned into pGEM-T Easy vector (Promega). Theloadings of the Ha-Ras mRNA levels were corrected by using a 28S probeas described in Ten Asbroek et al. (NAR 28, 1133-1138).

Biodistribution Studies

The animal experiments were approved by the ethical committee and areregistered under No. DNL19.

Tritium labeling of oligonucleotides was performed using the heatexchange method described by Graham et al. (Graham, M. J., Freier, S.M., Crooke, R. M., Ecker, D. J., Maslova, R. N., and Lesnik, E. A.(1993). Tritium labeling of antisense oligonucleotides was carried outby exchange with tritiated water. Nucleic Acids Res., 21: 3737-3743).The only two introduced differences to the protocol were that only 1 mgwas labeled per oligonucleotide and that the separation of free tritiumfrom the labeled oligonucleotide was done by 3× G10 30cm Sephadexcolumns (the columns were made using 10 ml plastic pipettes).Radioactivity in all samples was counted after dissolving the samples inUltima Gold (Packard) scintillation fluid, and using a scintillationcounter.

For the biodstribution studies, female nude mice (NMRI nu/nu, CharlesRiver Netherlands, Maastricht, The Netherlands) with 15PC3 and Miapacallxenografts were used. See the in vivo experiment section for furtherdetails.

Tissue distribution studies of tritiated oligonucleotides were performedaccording to Bijsterbosch et al. (Bijsterbosch, M. K., Manoharan, M.,Rump, E. T., De Vrueh, R. L., van Veghel, R., Tivel, K. L., Biessen, E.A., Bennett, C. F., Cook, P. D., and van Berkel T. J. (1997) In vivofate of phosphorothioate antisense oligodeoxynucleotides: predominantuptake by scavenger receptors on endothelial liver cells. Nucleic AcidsRes., 25: 3290-3296). The radioactivity in the different organs wascorrected for serum present at the time of sampling as determined by thedistribution of ¹²⁵I-BSA (personal communication K. Kruijt, Universityof Leiden, the Netherlands).

The oligonucleotides were either administrated by bolus injection in thelower vena cava (circulation for 30 minutes) or using Alzet osmoticminipumps (see in vivo experiment section), for a prolonged systemiccirculation. Tissue samples were dissolved in 5 M NaOH at 65° C. andsubsequently mixed with 10 volumes of Ultima Gold scintillation fluid.Serum and urine can be counted by mixing directly with Ultima gold.

In Vivo Experiment

The animal experiments were approved by the ethical committee and areregistered under No. DNL19. The detailed protocols of the animal studiesare described in two publications: Tumor genotype-specific growthinhibition in vivo by antisense oligonucleotides against a polymorphicsite of the large subunit of human RNA polymerase II. Fluiter K, tenAsbroek A L, van Groenigen M, Nooij M, Aalders M C, Baas F. Cancer Res2002 Apr. 1; 62(7):2024-2028

In vivo tumor growth inhibition and biodistribution studies of lockednucleic acid (LNA) antisense oligonucleotides. Fluiter K, ten Asbroek AL, de Wissel M B, Jakobs M E, Wissenbach M, Olsson H, Olsen 0, Oerum H,Baas F. Nucleic Acids Res 2003 Feb. 1; 31(3):953-962

Mice: Female NMRI nu/nu (Charles River Netherlands, Maastricht, TheNetherlands). Xenografts: MiaPaca II injected in the right flank s.c.with Matrigel (collaborative biomedical products Bedford, Mass.); 15PC3injected in the left flank s.c. with Matrigel. Osmotic pumps: Alzet 1002(DURECT Corporation, Cupertino, Calif.) lot no. 10045-02 Dosage for2776, 2778 (alpha-L-oxy-LNA), 2742 and 2744 (beta-D-oxy-LNA): 1 and 2.5mg/kg/day. Control: physiological saline.

Temperature and animal ID was monitored using: ELAM chips (IPTT 200)using a DAS 5002 chip reader (BMDS, Seaford, Del.).

Serum samples were taken for ASAT/ALAT and Alkaline Phosphatasedetermination. Aspartate aminotransferase (ASAT) and alanineaminotransferase (ALAT) levels and alkaline phosphatase in serum weredetermined using standard diagnostic procedures with the H747(Hitachi/Roche) with the appropriate kits (Roche Diagnostics). TheALAT/ASAT and Alkaline phosphatase Levels were determined approx 20hours post extraction of serum from the animal.

Results

Beta-D-Amino-LNA

Nuclease stability

One of the major difficulties encountered using the naturally occurringphosphodiester oligonucleotides as antisense probes is their rapiddegradation by various nucleolytic activities in cells, serum, tissuesor culture medium. Since the phosphorus center is the site ofnucleolytic attack, many modifications have been introduced in theinternucleoside linkage to prevent enzymatic degradation. To date, themost commonly employed synthetic modification is the backbonephosphorothioate analogue, made by replacing one of the non-bridgingoxygen atoms of the internucleoside linkage by sulfur.

We wanted to evaluate the effect of introducing the novel LNA within anoligonucleotide in the presence of nucleases, and to compare it with thewell-studied phosphorothioate oligonucleotides. The study was carriedout with oligothymidylates by blocking the 3′-end with the novel LNArelatives. The oligonucleotide is synthesized on deoxynucleoside-support(t).

From FIG. 1, we can appreciate the stability properties, which conferbeta-D-amino-LNA. Oligonucleotides containing T-monomer of2′-beta-D-amino-LNA (T^(N)) present a remarkable stability against a3′-exonuclease. Blocking the 3′-end with just two T^(N) stops the enzymefrom degrading the oligonucleotide at least for 2 h. See FIG. 1.

Assisted Cellular Uptake and Subcellular Distribution

The uptake efficiency of FAM-labeled oligonucleotide containingbeta-D-amino-LNA was measured as the mean fluorescence intensity of thetransfected cells by FACS analysis.

Two different transfection agents were tested (Lipofectamine 2000 andDAC30) in two different cell lines (MiaPacaII and 15PC3).

TABLE 1 Oligonucleotides (SEQ ID NOS 1-3, respectively, in orderof appearance) containing beta-D-amino-LNAused in cellular uptake and subcellular distributionexperiments. Residue c is metilyl-c both for DNA and LNA. DAC30Lipofectamine 2000 Ref oligonucleotides % cells % uptake % cells% uptake 2753T^(N)C^(N)C^(N)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(N)C^(N)T^(N)c-FAM— — 100 100 2752 T^(N) _(s)C^(N) _(s)C^(N)_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(N) _(s)C^(N)_(s)T^(N) _(s)c-FAM 30 30 100 100 2740T_(s)C_(s)C_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C_(s)C_(s)T_(s)c-FAM80 30 100 100

Oligonucleotides both fully thiolated (PS, 2752) and partially thiolated(PO in the flanks and PS in the gap, 2753) containing beta-D-amino-LNAlisted in table 1 were transfected with good efficiency, see table 1.Both transfection agents, DAC30 and Lipofectamine, presented goodtransfection efficiency; however, Lipofectamine was superior.

Lipofectamine showed 100% efficiency in all cases: for botholigonucleotides (2753 and 2752) and in both cell lines. Moreover, nosignificant differences in assisted transfection efficiency wereobserved between 2752 and 2753.

The FAM-labeled oligonucleotide 2752 was also used to assay thesubcellular distribution of 30 oligonucleotides containingbeta-D-amino-LNA, see FIG. 2. Most of the staining was detected asnuclear fluorescence that appeared as bright spherical structures (thenucleoli is also stained) in a diffuse nucleoplasmic background, as wellas some cytoplasmic staining in bright punctate structures. The observeddistribution patterns were similar for 15PC3 and MiaPacaII.

The subcellular distribution of beta-D-amino-LNA was comparable to theone observed with beta-D-oxy-LNA, 2740.

The uptake efficiency was also measured with tritium-labeledoligonucleotide 2754 (see table 3 and FIG. 3) at differentconcentrations 100, 200, 300 and 400 nM, using Lipofectamine2000 astransfection agent, both in MiaPacaII and 15PC3 cells, and compared withthe equivalent beta-D-oxy-LNA, 2742 (see table 3). 2754 shows loweruptake than 2742.

Antisense Activity Assay: Luciferase target

It has been shown that beta-D-oxy-LNA does not elicit RNaseH activity,which is the most common mode of action for an antisense oligonucleotidetargeting the down-stream region of the mRNA. However, this disadvantagecan be overcome by creating chimeric oligonucleotides composed ofbeta-D-oxy-LNA and a DNA gap positioned in the middle of the sequence. Agapmer is based on a central stretch of 4-12 DNA (gap) typically flankedby 1 to 6 residues of 2′-O modified nucleotides (beta-D-oxy-LNA in ourcase, flanks). It was of our interest to evaluate the antisense activityof oligonucleotides, which contain beta-D-amino-LNA in a gapmer design,and compare them with beta-D-oxy-LNA/DNA gapmers.

The oligonucleotides from table 2 were prepared. We decided to carry outthe study with gapmers of 16nt in length and a gap of 7nt, which contain4 residues of beta-D-amino-LNA in one flank and 4 residues ofbeta-D-oxy-LNA in the other flank, and a thiolated gap. The FAM groupwas shown not to affect the antisense ability of the oligonucleotides.Therefore, we prepared a FAM-labelled oligonucleotide to be both testedin the Luciferase assay, and in the Cellular uptake (unassisted).

The oligonucleotide, which targets a motif of the mRNA of the FireflyLuciferase, contains two mismatches in the flanks. Two C residues of the5′-end LNA flank were substituted for two Ts for synthetic reasons. Atthat point in time, only the T residues were available. Therefore and inorder to be able to establish a correct comparison, the correspondingbeta-D-oxy-LNA control was also included in the assay. No FAM labelingwas necessary in this case.

TABLE 2 Oligonucleotide (SEQ ID NOS. 4-5, respectively, in orderof appearance) containing beta-D-amino-LNA used in theantisense activity assay and the oxy-LNA control (Capitalletters for LNA and small letters for DNA, T^(N) isbeta-D-amino-LNA). Residue c is methyl-c both for LNA. ref sequencedesign size U-14FAM-T^(N)T^(N)T^(N)T^(N)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)TCTTTAmino-LNA in one flank/PS gap of 7 16 mer 2023-m;TTTTg_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)TCTTT Control with oxy-LNA 16 mer02579

From FIG. 4, we can see that the oligonucleotide with beta-D-amino-LNApresents good antisense activity at 50 nM oligonucleotide concentration.The inclusion of beta-D-amino-LNA in the flanks of an oligonucleotideresults in good down-regulation. We can conclude that the antisenseactivity of an oligonucleotide containing beta-D-amino-LNA is at leastas good as the parent all beta-D-oxy-LNA gapmer.

Antisense Activity Assay: Ha-Ras Target

It was of our interest to further evaluate the antisense activity ofoligonucleotides containing beta-D-amino-LNA in a gapmer design, andcompare them with beta-D-oxy-LNA gapmers.

The oligonucleotides from table 3 were prepared. We decided to carry outthe study with oligonucleotides of 16nt in length and a gap of 8nt,which contain 3 residues of beta-D-amino-LNA in each flank and adifferent extent of thiolation. 2754 is fully thiolated (PS), while 2755is only thiolated in the gap (PO in the flanks and PS in the gap). Theoligonucleotides were designed to target a motif of the mRNA of Ha-Ras.Different mismatch controls were also included, 2756 is fully thiolatedand 2757 presents thiolation only in the gap, see table 3. Moreover, thecorresponding beta-D-oxy-LNA gapmers (see table 3, 2742 is all PS, 2744is the corresponding mismatch control; 2743 has PS in the gap, 2745 isthe corresponding mismatch control) were also tested.

TABLE 3 Oligonucleotides (SEQ ID NOS 6-13, respectively,in order of appearance) containing beta-D-amino-LNA and beta-D-oxy-LNA used in the antisenseactivity experiments. Residue c is methyl-c both for DNA and LNA. refoligonucleotides 2755T^(N)C^(N)C^(N)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(N)C^(N)T^(N)cPO/PS 2754 T^(N) _(s)C^(N) _(s)C^(N)_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(N) _(s)C^(N)_(s)T^(N) _(s)c All PS 2743TCCg_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)CCTc PO/PS 2742T_(s)C_(s)C_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C_(s)C_(s)T_(s)cAll PS 2757T^(N)C^(N)T^(N)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C^(N)C^(N)C^(N)cMismatch control 2756 T^(N) _(s)C^(N) _(s)T^(N)_(s)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C^(N) _(s)C^(N)_(s)C^(N) _(s)c Mismatch control 2745TCTg_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)CCCc Mismatch control2744T_(s)C_(s)T_(s)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C_(s)C_(s)C_(s)cMismatch control

The Ras family of mammalian proto-oncogenes includes three well-knownisoforms termed Ha-Ras (Ha-Ras), Ki-Ras (K-Ras) and N-Ras. The rasproto-oncogenes encode a group of plasma membrane associated G-proteinsthat bind guanine nucleotides with high affinity and activates severaleffectors including raf-1, PI3-K etc. that are known to activate severaldistinct signaling cascades involved in the regulation of cellularsurvival, proliferation and differentiation.

Several in vitro (and in vivo) studies have demonstrated that the Rasfamily of proto-oncogenes are involved in the induction of malignanttransformation. Consequently, the Ras family is regarded as importanttargets in development of anticancer drugs, and it has been found thatthe Ras proteins are either over-expressed or mutated (often leading toconstitutive active Ras proteins) in approximately 25% of all humancancers.

Interestingly, the ras gene mutations in most cancer types arefrequently limited to only one of the ras genes and are dependent ontumor type and tissue. Mutations in the Ha-Ras gene are mainlyrestricted to urinary tract and bladder cancer.

The inclusion of beta-D-amino-LNA in the flanks of an oligonucleotideresults in good down-regulation levels. From FIG. 5, we can see thatoligonucleotides with beta-D-amino-LNA present good antisense activityat two different concentrations, 400 and 800 nM. No significantdifference in down-regulation can be seen between oligonucleotides 2755and 2754, which present a different degree in thiolation. We canconclude that the antisense activity of an oligonucleotide containingbeta-D-amino-LNA is at least as good as the parent beta-D-oxy-LNAgapmer. From FIG. 6, a wider range of concentration was tested. There isa potent down-regulation between 50-400 nM for 2754. The specificity wasalso tested; at 30 nM there is a significant difference indown-regulation between the mismatch 2756 (less potent) and the match2754. Lower concentrations (5-40 nM) were also included from the tablein FIG. 6. Potent down-regulation is observed even at 5 nM for 2754, andthese levels of down-regulation are comparable to the correspondingbeta-D-LNA control, 2742. The specificity is also remarkable, if wecompare the antisense activity for 2754 at 20 nM (8.7% down-regulation)in comparison with the mismatch containing control 2756 (56.2%down-regulation).

Biodistribution

The biodistribution of oligonucleotides containing beta-D-amino-LNA(tritiated 2754) was also studied, both after i.v. injection and usingAlzet osmotic minipumps. 2754 was administered to xenografted mice with15PC3 tumors on the left side and MiaPacaII tumors on the right side asan intraveneous injection, and the analysis was carried out after 30 mincirculation. From FIG. 7, the serum clearance for 2754 is very rapid,and the biodistribution looks very similar to the biodistributionpattern presented by the reference containing beta-D-oxy-LNA; the kidneyand the liver (to lesser extent) are the main sites of uptake, whencorrected for tissue weight.

Moreover, a group of 4 nude mice xenografted with 15PC3 tumors on theleft side and MiaPacaII tumors on the right side were treated for 72 hwith Alzet osmotic minipumps with a 2.5 mg/Kg/day dosage of tritiated2754. After the treatment, the radioactivity present in the differenttissues was measured. FIG. 8 shows the distribution of 2754 in thetissues as a total uptake and as a specific uptake. It seems that thetissue takes up significantly better amino-LNA than beta-D-oxy LNA. Themain sites of uptake were liver, muscle, kidney, skin, bone and heart.When corrected for tissue weight, kidney, heart and liver (lungs andmuscle in a lower extent) were the main uptake sites. This patterndiffers to a certain extent from the one observed for beta-D-oxy-LNA. Itis also noteworthy that the uptake of amino-LNA is significantly betterin tumor tissue than for e.g. beta-D-oxy LNA (see FIGS. 7 and 8).

RNase H Assay

Rnase H is a ubiquitous cellular enzyme that specifically degrades theRNA strand of DNA/RNA hybrids, and thereby inactivates the mRNA towardfurther cellular metabolic processes. The inhibitory potency of someantisense agents seems to correlate with their ability to elicitribonuclease H (RNaseH) degradation of the RNA target, which isconsidered 20 a potent mode of action of antisense oligonucleotides. Assuch, understanding the mechanisms of catalytic function and substraterecognition for the RNaseH is critical in the design of potentialantisense molecules.

It was our aim to evaluate the RNaseH activity of gapmers containingbeta-D-amino-LNA. From FIG. 9, we can appreciate a good cleavageactivity for an oligonucleotide containing 25 beta-D-amino-LNA, as intable 2.

Beta-D-Thio-LNA

Nuclease Stability

As we did for beta-D-amino-LNA, beta-D-thio-LNA was also evaluatedagainst a 3′-exonuclease (SVPD). The oligonucleotide is synthesized ondeoxynucleoside-support (t). The study was carried out witholigothymidylates by blocking the 3′-end with beta-D-thio-LNA.

From FIG. 10, we can see that the incorporation of just one T-monomer of2′-beta-D-thio-LNA (Ts) has a significant effect in the nucleolyticresistance of the oligonucleotide towards SVPD. After 2 h digestion morethan 80% of the oligonucleotide remains, while the correspondingbeta-D-oxy-LNA oligonucleotide is digested by the exonuclease, see FIG.10.

Unassisted Cellular Uptake

The efficiency of FAM-labelled oligonucleotide uptake was measured asthe mean fluorescence intensity of the transfected cells by FACSanalysis.

The transfection without lipid showed distinct differences between thetested 5 oligonucleotides. The uptake as measured from mean fluorescenceintensity of transfected cells was dose dependent.

Gapmers (16nt in length and gap of 7nt) containing beta-D-thio-LNA inthe flanks were analysed and compared with the correspondingbeta-D-oxy-LNA gapmers. Beta-D-thio-LNA (one flank with beta-D-thio-LNAand the other one with oxy-LNA, as in table 5) showed higher uptake thanoligonucleotides containing only oxy-LNA. The beta-D-thio-LNAoligonucleotides (both all-PO gapmer and gapmer with PS-gap andPO-flanks) had good uptake efficiency. Specially, the all-PO gapmercontaining beta-D-thio-LNA was far superior to other all-POoligonucleotides tested so far, as it can be appreciated from FIG. 11.

Assisted Cellular Uptake and Subcellular Distribution

The uptake efficiency of FAM-labeled oligonucleotide containingbeta-D-thio-LNA was measured as the mean fluorescence intensity of thetransfected cells by FACS analysis. Two different transfection agentswere tested (Lipofectamine 2000 and DAC30) in two different cell lines(MiaPacall and 15PC3).

TABLE 4 Oligonucleotides (SEQ ID NOS 14-15 & 3, respectively, in orderof appearance) containing beta-D-thio-LNA used in cellularuptake and subcellular distribution experiments.Residue c is methyl-c both for DNA and LNA. DAC30 Lipofectamine 2000 refoligonucleotides % cells % uptake % cells % uptake 2747T^(S)C^(S)C^(S)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(S)C^(S)T^(S)c-FAM— — 100 100 2746 T^(S) _(s)C^(S) _(s)C^(S)_(s)g_(s)t_(s)c_(s)a_(s)t_(s)C_(s)g_(s)c_(s)t_(s)C^(S) _(s)C^(S)_(s)T^(S) _(s)c-FAM 80 50 100 100 2740T_(s)C_(s)C_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C_(s)C_(s)T_(s)c-FAM80 30 100 100

Oligonucleotides both fully thiolated (PS, 2746) and partially thiolated(PO in the flanks and PS in the gap, 2747) containing beta-D-thio-LNAlisted in table 4 were transfected with good efficiency, see table 4.Both transfection agents, DAC30 and Lipofectamine, presented goodtransfection efficiency; however, Lipofectamine was superior.Lipofectamine showed 100% efficiency in all cases: for botholigonucleotides (2746 and 2747) and in both cell lines. Moreover, nosignificant differences in assisted transfection efficiency wereobserved between 2746 and 2747.

The FAM-labeled oligonucleotide 2746 was also used to assay thesubcellular distribution of oligonucleotides containing beta-D-thio-LNA,see FIG. 2. Most of the staining was detected as nuclear fluorescencethat appeared as bright spherical structures (the nucleoli is alsostained) in a diffuse nucleoplasmic background, as well as somecytoplasmic staining in bright punctate structures. The observeddistribution patterns were similar for 15PC3 and MiaPacall.

The subcellular distribution of beta-D-thio-LNA was comparable to theone observed with beta-D-oxy-LNA, 2740.

The uptake efficiency was also measured with tritium-labeledoligonucleotide 2748 (see table 6 and FIG. 3) at differentconcentrations 100, 200, 300 and 400 nM, using Lipofectamine2000 astransfection agent, both in MiaPacall and 15PC3 cells, and compared withthe equivalent beta-D-oxy-LNA, 2742 (see table 6). 2748 shows superioruptake than 2742.

Antisense Activity Assay: Luciferase Target

We also introduced beta-D-thio-LNA in a gapmer design, and evaluated itin terms of antisense activity.

The oligonucleotides from table 5 were prepared. We decided to carry outthe study with gapmers of 16nt in length and a gap of 7nt, which contain4 residues of beta-D-thio-LNA in one flank and 4 residues of oxy-LNA inthe other flank, and a thiolated gap.

The FAM group was shown not to affect the antisense ability of theoligonucleotides. Therefore, we prepared a FAM-labelled oligonucleotideto be both tested in the Luciferase assay, and in the Cellular uptake(unassisted).

The oligonucleotide, which is directed against a motif of the mRNA ofthe firefly luciferase, contains two mismatches in the flanks. Two Cresidues of the 5′-end LNA flank were substituted for two Ts forsynthetic reasons. At that point in time, only the T residues wereavailable. Therefore and in order to be able to establish a correctcomparison, the corresponding oxy-LNA control was also included in theassay. No FAM labeling was necessary in this case.

TABLE 5 Oligonucleotide (SEQ ID NOS 16 & 5, respectively, in orderof appearance containing beta-D-thio-LNA used in the antisenseactivity assay and the corresponding oxy-LNA control (Capitalletters for LNA and small letters for DNA, T^(S) is beta-D-thio-LNA).Residue c is methyl-c both for LNA. ref sequence design size U-16T^(S)T^(S)T^(S)T^(S)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)TCTTT-FAMThio-LNA in one flank/PS gap of 7 16 mer 2023-m;TTTTg_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)TCTTT Control with oxy-LNA 16 mer02579

From FIG. 4, it can be seen that the oligonucleotide withbeta-D-thio-LNA presents good antisense activity at 50 nMoligonucleotide concentration. Therefore, the inclusion ofbeta-D-thio-LNA in the flanks of an oligonucleotide results in gooddown-regulation, and is at least as good as the parent allbeta-D-oxy-LNA gapmer.

Antisense Activity Assay: Ha-Ras Target

It was of our interest to further evaluate the antisense activity ofoligonucleotides containing beta-D-thio-LNA in a gapmer design, andcompare them with beta-D-oxy-LNA gapmers.

The oligonucleotides from table 6 were prepared. We decided to carry outthe study with oligonucleotides of 16nt in length and a gap of 8nt,which contain 3 residues of beta-D-thio-LNA in each flank and adifferent extent of thiolation. 2748 is fully thiolated (PS), while 2749is only thiolated in the gap (PO in the flanks and PS in the gap). Theoligonucleotides were designed to target a motif of the mRNA of Ha-Ras.Different mismatch controls were also included, 2750 is fully thiolatedand 2751 presents thiolation only in the gap, see table 6. Moreover, thecorresponding beta-D-oxy-LNA gapmers (see table 6, 2742 is all PS, 2744is the corresponding mismatch control; 2743 has PS in the gap, 2745 isthe corresponding mismatch control) were also tested.

TABLE 6 Oligonucleotides (SEQ ID NOS 17-18, 8-9, 19-20& 12-13, respectively, in order of appearance)containing beta-D-thio-LNA and beta-D-oxy-LNAused in the antisense activity experiments.Residue c is methyl-c both for DNA and LNA. ref oligonucleotides 2749T^(S)C^(S)C^(S)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(S)C^(S)T^(S)cPO/PS 2748 T^(S) _(s)C^(S) _(s)C^(S)_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(S) _(s)C^(S)_(s)T^(S) _(s)c All PS 2743TCCg_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)CCTc PO/PS 2742T_(s)C_(s)C_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C_(s)C_(s)T_(s)cAll PS 2751T^(S)C^(S)T^(S)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C^(S)C^(S)C^(S)cMismatch control 2750 T^(S) _(s)C^(S) _(s)T^(S)_(s)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C^(S) _(s)C^(S)_(s)C^(S) _(s)c Mismatch control 2745TCTg_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)CCCc Mismatch control2744T_(s)C_(s)T_(s)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C_(s)C_(s)C_(s)cMismatch control

The inclusion of beta-D-thio-LNA in the flanks of an oligonucleotideresults in good down-regulation levels. From FIG. 5, we can see thatoligonucleotides with beta-D-thio-LNA present good antisense activity attwo different concentrations, 400 and 800 nM. No significant differencein down-regulation can be seen between oligonucleotides 2749 and 2748,which present a different degree in thiolation. However, 2749 presentsbetter levels of down-regulation, both at 400 and 800 nM. We canconclude that the antisense activity of an oligonucleotide containingbeta-D-thio-LNA lies in the range of the parent beta-D-oxy-LNA gapmer.From FIG. 6, a wider range of concentration was tested. There is apotent down-regulation between 50-400 nM for 2748. The specificity wasalso tested; at 30 nM there is a significant difference indown-regulation between the mismatch 2750 (less potent) and the match2748.

Biodistribution

The biodistribution of oligonucleotides containing beta-D-thio-LNA(tritiated 2748) was also studied, both after i.v. injection and usingAlzet osmotic minipumps.

2748 was administered to xenografted mice with 15PC3 tumors on the leftside and MiaPacaII tumors on the right side as an intraveneousinjection, and the analysis was carried out after 30 min circulation.From FIG. 7, the serum clearance for 2748 is very rapid, and thebiodistribution looks very similar to the biodistribution patternpresented by the reference containing beta-D-oxy-LNA; the kidney and theliver (to lesser extent) are the main sites of uptake, when correctedfor tissue weight.

Moreover, a group of 4 nude mice xenografted with 15PC3 tumors on theleft side and MiaPacaII tumors on the right side were treated for 72 hwith Alzet osmotic minipumps with a 2.5 mg/Kg/day dosage. After thetreatment, the radioactivity present in the different tissues wasmeasured. FIG. 8 shows the distribution of 2748 in the tissues as atotal uptake and as a specific uptake. The main sites of uptake wereliver, muscle, kidney, skin and bone. When corrected for tissue weight,kidney and liver were the main uptake sites.

RNaseH Assay

We also evaluated gapmer designs that contain beta-D-thio-LNA, as intable 5, for their ability to recruit RNaseH activity.

From FIG. 9, we can see that a beta-D-thio-LNA gapmer recruits RnaseHactivity.

Alpha-L-oxy LNA

Nuclease Stability

The stabilization properties of alpha-L-oxy-LNA were also evaluated. Thestudy was carried out with oligothymidylates by blocking the 3′-end withalpha-L-oxy-LNA. The oligonucleotide is synthesized ondeoxynucleoside-support (t). From FIG. 12, we can see that theintroduction of just one alpha-L-T (T^(α)) at the 3′-end of theoligonucleotide represents already a gain of 40% stability (after 2 hdigestion) with respect to the oxy-version, for which there was actuallyno gain. The addition of two modifications contributes even more to thestability of the oligonucleotide.

Furthermore, we investigated the effect on stability againstS1-endonuclease of alpha-L-oxy-LNA for a 16mer fully modifiedoligothymidylates. The increased stability of these modifiedoligonucleotides relative to their deoxynucleotide and phosphorothioatebackbone relatives was compared in order to carefully assess thecontribution of the alpha-L-oxy-LNA modification.

After 2 h digestion, most of the alpha-L-oxy-LNA oligonucleotideremained (over 80% of the full-length product remained), while neitherthe oligodeoxynucleotide nor the DNA phosphorothioate analogue could bedetected after 30 min digestion (see FIG. 13). The same kinetic studyagainst S1-endonuclease was carried out with a fully modified oxy-LNAoligonucleotide, which was also very resistant against theS1-endonuclease. Over an 85% of the full-length product remained after 2h digestion (see FIG. 13).

In conclusion, beta-D-oxy-LNA, beta-D-amino-LNA, beta-D-thio-LNA andalpha-L-oxy-LNA stabilize oligonucleotides against nucleases. An orderof efficiency in stabilization can be established: DNAphosphorothioates<<oxy-LNA<α-L-oxy-LNA<beta-D-amino-LNA<beta-D-thio-LNA.

Unassisted Cellular Uptake

The efficiency of FAM-labelled oligonucleotide uptake was measured asthe mean fluorescence intensity of the transfected cells by FACSanalysis. The uptake as measured from mean fluorescence intensity oftransfected cells was dose dependent. Gapmers (16nt in length and gap of7nt) containing α-L-oxy-LNA in the flanks were analysed and comparedwith the corresponding beta-D-oxy-LNA gapmer. α-L-oxy-LNA (in bothflanks) showed higher uptake than the oligonucleotide containing onlybeta-D-oxy-LNA. Both all-PO and gapmer with PS-gap had good uptakeefficiency; especially the all-PO gapmer was far superior than other allPO oligonucleotides tested so far, see FIG. 14 for FACS analysis.

Assisted Cellular Uptake and Subcellular Distribution

The uptake efficiency of FAM-labeled oligonucleotides containingalpha-L-oxy-LNA was measured as the mean fluorescence intensity of thetransfected cells by FACS analysis. Two different transfection agentswere tested (Lipofectamine 2000 and DAC30) in two different cancer celllines (MiaPacaII and 15PC3).

TABLE 7 Oligonucleotides (SEQ ID NOS 21-22 & 3, respectively in orderof appearance) containing alpha-L-oxy-LNA used in cellular uptakeand subcellular distribution experiments. Residue c ismethyl-c both for DNA and LNA. DAC30 Lipofectamine 2000 refoligonucleotides % cells % uptake % cells % uptake 2773T^(a)C^(a)C^(a)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(a)C^(a)T^(a)c-FAM— — 100 100 2774 T^(a) _(s)C^(a) _(s)C^(a)_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(a) _(s)C^(a)_(s)T^(a) _(s)c-FAM 80 30 100 100 2740T_(s)C_(s)C_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C_(s)C_(s)T_(s)c-FAM80 30 100 100

Oligonucleotides both fully thiolated (PS, 2774) and partially thiolated(PO in the flanks and PS in the gap, 2773) containing alpha-L-oxy-LNAlisted in table 7 were transfected with good efficiency, see table 7.Both transfection agents, DAC30 and Lipofectamine, presented goodtransfection efficiency; however, Lipofectamine was superior.

Lipofectamine showed 100% efficiency in all cases: for botholigonucleotides (2773 and 2774) and in both cell lines. Moreover, nosignificant differences in assisted transfection efficiency wereobserved between 2773 and 2774.

The FAM-labeled oligonucleotide 2774 was also used to assay thesubcellular distribution of oligonucleotides containing alpha-L-oxy-LNA,see FIG. 2. Most of the staining was detected as nuclear fluorescencethat appeared as bright spherical structures (the nucleoli is alsostained) in a diffuse nucleoplasmic background, as well as somecytoplasmic staining in bright punctate structures. The observeddistribution patterns were similar for 15PC3 and MiaPacaII.

The subcellular distribution of alpha-L-oxy-LNA was comparable to theone observed with beta-D-oxy-LNA, 2740.

Antisense Activity: Luciferase Target

Gapmers Containing Alpha-L-oxy-LNA

We also wanted to see the antisense acticvity in a gapmeroligonucleotides containing alpha-L-oxy-LNA (16nt in length with athiolated 7nt gap). Two different designs were evaluated.

First, we substituted two oxy-LNA residues for two alpha-L-oxy-LNAs in agapmer against a motif of the mRNA of the firefly luciferase, and placedthe alpha-L-oxy-LNA in the junctions, see FIG. 15.

Then, we substituted both flanks with alpha-L-oxy-LNA in the sameconstruct, see FIG. 15.

Previously, different oligonucleotides were tested and compared with thecorresponding FAM-labelled molecules, and no significant difference wasappreciated between the free and FAM-labelled ones. Therefore, weincluded oligonucleotides from the Unassisted Cellular Uptake assay inthe Luciferase assay study, assuming that the antisense activity willnot be affected by the presence of the FAM group.

From FIG. 16, the oligonucleotide with alpha-L-oxy-LNA in the junctionsshows potent antisense activity. It is actually 5-fold better than thecorresponding all oxy-LNA gapmer (gap of 7nt), and slightly better thana gapmer with an optimised 9nt gap with oxy-LNA.

The second design (all alpha-L-oxy-LNAs in both flanks) presents atleast as good down-regulation levels as the observed for beta-D-oxy-LNAgapmers. We can also conclude that the presence of the alpha-L-oxy-LNAin a gapmer construct shows good-antisense activity level.

alpha-L-oxy-LNA reveals to be a potent tool enabling the construction ofdifferent gapmers, which show good antisense activity. The placement ofalpha-L-oxy-LNA in the junctions results in a very potentoligonucleotide.

Short-Sized Gapmers Containing Alpha-L-oxy-LNA

As a general rule, the length of the construct is usually designed torange from 15-25 nucleotide units, in order to ensure that optimalidentification and binding takes place with a unique sequence in themammalian genome and not with similar genetically redundant elements.Statistical analyses specify 11-15 base paired human sequences as thetheoretical lower limits for sufficient recognition of a single genomicregion. In practice, however, a longer oligonucleotide is commonly usedto compensate for low melting transitions, especially for thiolatedoligonucleotides that have lower affinity.

As a significant increase in affinity is achieved by the introduction ofoxy-LNA or novel LNA relatives, the design of potent and short antisenseoligonucleotides (<15nt) should be enabled.

The alpha-L-oxy-LNA can play an important role in enabling the design ofshort molecules by maintaining the required high-affinity, but also anoptimal gap size. 12 and 14 mers against a motif of the mRNA of thefirefly luciferase were evaluated.

The results are shown in FIG. 16. The presence of alpha-L-oxy-LNA in theflanks of a 12 (gap of 7nt) and 14 mer (gap of 8nt) correspond to goodlevels of down-regulation. From FIG. 16.

In conclusion, alpha-L-oxy-LNA is a potent tool in enabling the designof short antisense oligonucleotides with significant down-regulationlevels.

Mixmers Containing Alpha-L-oxy-LNA

We also considered other designs containing alpha-L-oxy-LNA against amotif of the mRNA of the firefly luciferase, which we called mixmers.They consist of an alternate composition of DNA, alpha-L-oxy-LNA andbeta-D-oxy-LNA. The following figure illustrates the chosen designs. Wenamed the mixmers by the alternate number of units of eachalpha-L-oxy-LNA, beta-D-oxy-LNA or DNA composition. See FIG. 17 andtable 8 for the different designs.

TABLE 8 Mixmers (SEQ ID NOS 23-26, respectively, inorder of appearance) containing alpha-L-oxy-LNAused in this study (Capital letters for LNAand small letters for DNA, T″ is alpha-L-oxy-LNA).Residue c is methyl-c both for LNA. ref sequence mixmer 2023-qTTCCg_(s)T^(a) _(s)c_(s)a_(s)t_(s)c_(s)g_(s)T^(a) _(s)c_(s)TTT4-1-1-5-1-1-3 a 2023-r T^(a)T^(a)C^(a)C^(a)g_(s)T^(a)_(s)c_(s)a_(s)t_(s)c_(s)g_(s)T^(a) _(s)c_(s)T^(a)T^(a)T 4-1-1-5-1-1-3 b2023-t TTCCg_(s)t_(s)c_(s)A^(a) _(s)t_(s)c_(s)g_(s)TCTTT 4-3-1-3-5 a2023-u TTCC^(a)g_(s)t_(s)c_(s)A^(a) _(s)t_(s)c_(s)g_(s)T^(a)CTTT4-3-1-3-5 b

In design 4-1-1-5-1-1-3 (FIG. 17, table 8), we placed twoalpha-L-oxy-LNA residues interrupting the gap, being the flanksbeta-D-oxy-LNA. Furthermore, we interrupted the gap with twoalpha-L-oxy-LNA residues, and substituted both flanks withalpha-L-oxy-LNA. The presence of alpha-L-oxy-LNA might introduce aflexible transition between the North-locked flanks (oxy-LNA) and thealpha-L-oxy-LNA residue by spiking in deoxynucleotide residues.

It is also interesting to study design 4-3-1-3-5 (FIG. 17, table 8),where an alpha-L-oxy-LNA residue interrupts the DNA stretch. In additionto the alpha-L-oxy-LNA in the gap, we also substituted two oxy-LNAresidues at the edges of the flanks with two alpha-L-oxy-LNA residues.

The presence of just one beta-D-oxy-LNA residue (design 4-3-1-3-5)interrupting the stretch of DNAs in the gap results in a dramatic lossof down-regulation. Just by using alpha-L-oxy-LNA instead, the designshows significant down-regulation at 50 nM oligonucleotideconcentration, see FIG. 16. The placement of alpha-L-oxy-LNA in thejunctions and one alpha-L-oxy-LNA in the middle of the gap also showsdown-regulation, see FIG. 16.

The interruption of the gap with two beta-D-oxy-LNAs (design4-1-1-5-1-1-3) relates also with a loss in antisense activity. Again thefully substitution of beta-D-oxy-LNA for alpha-L-oxy-LNA givessignificant antisense activity, see FIG. 16. alpha-L-oxy-LNA reveals tobe a potent tool enabling the construction of different mixmers, whichare able to present high levels of antisense activity.

Other Designs

Other mixmers containing alpha-L-oxy-LNA were studied, see FIG. 18.Furthermore, mixmers, such as in table 8 and FIG. 17, but with nothiolation, were also tested.

Antisense Activity Assay: Ha-Ras Target

It was of our interest to further evaluate the antisense activity ofoligonucleotides containing alpha-L-oxy-LNA in a gapmer design, andcompare them with beta-D-oxy-LNA gapmers.

The oligonucleotides from table 9 were prepared. We decided to carry outthe study with oligonucleotides of 16nt in length and a gap of 8nt,which contain 3 residues of alpha-L-oxy-LNA in each flank and adifferent extent of thiolation. 2776 is fully thiolated (PS), while 2775is only thiolated in the gap (PO in the flanks and PS in the gap). Theoligonucleotides were designed to target a motif of the mRNA of Ha-Ras.Different mismatch controls were also included, 2778 is fully thiolatedand 2777 presents thiolation only in the gap, see table 9. Moreover, thecorresponding beta-D-oxy-LNA gapmers (see table 9, 2742 is all PS, 2744is the corresponding mismatch control; 2743 has PS in the gap, 2745 isthe corresponding mismatch control) were also tested.

TABLE 9 Oligonucleotides (SEQ ID NOS 27-28, 8-9, 29-30 &12-13, respectively, in order of appearance),containing alpha-L-oxy-LNA and beta-D-oxy-LNAused in the antisense activity experiments.Residue c is methyi-c both for DNA and LNA. ref oligonucleotides 2775T^(a)C^(a)C^(a)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(a)C^(a)T^(a)cPO/PS 2776 T^(a) _(s)C^(a) _(s)C^(a)_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(a) _(s)C^(a)_(s)T^(a) _(s)c All PS 2743TCCg_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)CCTc PO/PS 2742T_(s)C_(s)C_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C_(s)C_(s)T_(s)cAll PS 2777T^(a)C^(a)T^(a)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C^(a)C^(a)C^(a)cMismatch control 2778 T^(a) _(s)C^(a) _(s)T^(a)_(s)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)C_(s)c_(s)C^(a) _(s)C^(a)_(s)C^(a) _(s)c Mismatch control 2745TCTg_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)C_(s)c_(s)CCCc Mismatch control2744T_(s)C_(s)T_(s)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C_(s)C_(s)C_(s)cMismatch control

The inclusion of alpha-L-oxy-LNA in the flanks of an oligonucleotideresults in good down-regulation levels. From FIG. 6, we can see that theoligonucleotide 2776 with alpha-L-oxy-LNA present good antisenseactivity at a different range of concentrations, 50 nM-400 nM. Nosignificant difference in down-regulation can be seen between 2776 and2742. We can conclude that the antisense activity of an oligonucleotidecontaining alpha-L-oxy-LNA is at least as good as the parentbeta-D-oxy-LNA gapmer. The specificity was also tested; at 30 nM thereis a significant difference in down-regulation between the mismatch 2778(less potent) and the match 2776. Lower concentrations (5-40 nM) werealso included from the table in FIG. 6. Potent down-regulation isobserved even at 5 nM for 2776 in comparison with the correspondingbeta-D-oxy-LNA control, 2742. The specificity is also remarkable, if wecompare the antisense activity for 2776 at 20 nM (2.6% down-regulation)in comparison with the mismatch containing control 2778 (77%down-regulation).

RNaseH Assay

We also evaluated gapmer designs that contain alpha-L-oxy-LNA for theirability to recruit RNaseH activity.

alpha-L-oxy-LNA gapmer and mixmer designs recruit RnaseH activity, seeFIG. 19.

In Vivo Experiment

Nude mice were injected s.c. with MiaPaca II cells (right flank) and15PC3 cells (left flank) 10 one week prior to the start ofoligonucleotide treatment to allow xenograft growth. The anti Ha-Rasoligonucleotides (2742 and 2776, table 10) and control oligonucleotides(2744 and 2778, table 10) were administrated for 14 days using Alzetosmotic minipumps (model 1002) implanted dorsally. Two dosages wereused: 1 and 2.5 mg/Kg/day. During treatment the tumor growth wasmonitored. Tumor growth was almost inhibited completely at 2.5 mg/Kg/dayand even at 1 mg/Kg/day dose with 2742 and 2776 in 15PC3 cells, FIG. 20.The specificity with control oligonucleotides (2744 and 2778, containingmismatches) increased as the dose decreased. At 1 mg/Kg/day dose theexperiment presented a good specificity, particularly foralpha-L-oxy-LNA oligonucleotides (2742 and 2744). In MiaPacaII xenografttumors, the effect of the oligonucleotides is in general comparable withthose on the 15PC3 xenografts, except for the fact that the specificityseemed to be a bit lower. It can be concluded that the oligonucleotidecontaining alpha-L-oxy-LNA are as potent, or maybe even better, as theone containing beta-D-oxy-LNA in tumor growth inhibition in theconcentration range tested.

TABLE 10 Oligonucleotides. (SEQ ID NOS 28, 30, 9 & 13,respectively, in order of appearance)containing alpha-L-oxy-LNA and beta-D-oxy-LNAused in the in vivo experiments. Residuec is methyl-c both for DNA and LNA. ref oligonucleotides 2776 T^(a)_(s)C^(a) _(s)C^(a)_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C^(a) _(s)C^(a)_(s)T^(a) _(s)c match 2778 T^(a) _(s)C^(a) _(s)T^(a)_(s)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C^(a) _(s)C^(a)_(s)C^(a)a_(s)c Mismatch control 2742T_(s)C_(s)C_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)c_(s)t_(s)C_(s)C_(s)T_(s)cmatch 2744T_(s)C_(s)T_(s)g_(s)t_(s)a_(s)a_(s)t_(s)a_(s)g_(s)c_(s)c_(s)C_(s)C_(s)C_(s)cMismatch control

Toxicity Levels

The levels of aspartate aminotransferase (ASAT), alanineaminotransferase (ALAT) and alkaline phosphatase in the serum weredetermined, in order to study the possible effects of this 14-daytreatment in the nude mice. Serum samples were taken from each mouseafter the 14-day experiment. From FIG. 21, ALAT levels in the serumvaried between 250-500 U/L. ASAT levels were in the range of 80-150 U/L.The mice did not seem externally to be sick, and no big changes inbehavior were observed. During treatment the body temperature of themice was also monitored (FIG. 22). The body temperature did not changesignificantly during the treatment, not even at high dose 2.5 mg/Kg/day,which 5 is an indication that no major toxicity effects are occurring.In some cases, the body temperature of the mice was a bit higher,divided in two groups. These effects cannot be explained by the fact ofone oligonucleotide behaving differently or one dosage being too high.

Specific Beta-D-oxy-LNA Constructs

Luciferase Target: Antisense Activity Assay

Design 3-9-3-1 has a deoxynucleoside residue at the 3′-end, see table 11and FIG. 23. It shows significant levels of down-regulation, in the samerange than an optimised (9nt) fully thiolated gapmer. Moreover, onlypartial thiolation is needed for these mixmers to work as good as thefully thiolated gapmer, see FIG. 24.

TABLE 11 Special beta-D-oxy-LNA constructs (SEQ ID NOS31-33, respectively, in order of appearance)(Capital letters for LNA and small letters forDNA). Residue c is methyl-c for LNA. ref sequence mixmer 2023-l; 02574TTCc_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)t_(s)CTTt 3-9-3-12023-k; 02575 TTCc_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)t_(s)CTT_(s)t3-9-3-1 2023-j; 02576T_(s)T_(s)C_(s)c_(s)g_(s)t_(s)c_(s)a_(s)t_(s)c_(s)g_(s)t_(s)C_(s)T_(s)T_(s)t3-9-3-1

Other oligonucleotides containing novel LNA monomers (beta-D-amino-,beta-D-thio- and alpha-L-LNA) and bearing a deoxynucleoside residue atthe 3′-end were tested in different assays, see tables 3, 6, 9 and 10for more detail.

1.-42. (canceled)
 43. A gapmer oligonucleotide 12-26 nucleotide units inlength of formula A-B-C-D, wherein: A represents a sequence of lockednucleotide units; B represents a sequence of at least 6 continuous DNAnucleotide units; C represent a locked nucleotide unit or a sequence oflocked nucleotide units; D represents a non-locked nucleotide unit or asequence of non-locked nucleotide units, wherein the non-lockednucleotide unit of region D is a 2′ substituted non-locked nucleotide,wherein the 2′ substituent is selected from the group consisting ofhalogen or C₁-C₉ alkoxy.
 44. The gapmer oligonucleotide according toclaim 43, wherein at least one of the locked nucleotide units of regionsA and/or C is a nucleotide of formula:

wherein X is O; one of the H in (CH₂)n is replaced with a C₁₋₆ alkylsubstituent; n is 1; and Base is independently selected from the groupconsisting of cytosine, methyl cytosine, uracil, thymine, adenine andguanine.
 45. The gapmer oligonucleotide according to claim 44, whereinone of the H in (CH₂)n is replaced with methyl.
 46. The gapmeroligonucleotide according to claim 43 wherein at least one of theinternucleoside linkages is a phosphorothioate linkage.
 47. The gapmeroligonucleotide of claim 43 wherein all of the internucleotide linkagesare phosphorothioate linkages.
 48. The gapmer oligonucleotide accordingto claim 44 wherein at least one of the internucleotide linkages is aphosphorothioate linkages.
 49. The gapmer oligonucleotide according toclaim 44 wherein all of the internucleotide linkages arephosphorothioate linkages.
 50. The gapmer oligonucleotide of claim 43wherein in the central region is 8-12 DNA nucleotides.
 51. The gapmeroligonucleotide of claim 44 wherein in the central region is 8-12 DNAnucleotides.
 52. The gapmer oligonucleotide of claim 43, wherein regionA has a length of 2-6 nucleotide units and region C has a length of 1-5nucleotide units and D has a length of 1-3 nucleotide units.
 53. Thegapmer oligonucleotide of claim 44, wherein region A has a length of 2-6nucleotide units and region C has a length of 1-5 nucleotide units and Dhas a length of 1-3 nucleotide units.
 54. The gapmer oligonucleotide ofclaim 43, wherein region A has a length of 3-5 nucleotide units andregion C has a length of 2-4 nucleotide units and D has a length of 1-2nucleotide units.
 55. The gapmer oligonucleotide of claim 44, whereinregion A has a length of 3-5 nucleotide units and region C has a lengthof 2-4 nucleotide units and D has a length of 1-2 nucleotide units. 56.The gapmer oligonucleotide of claim 43, wherein the gapmeroligonucleotide has a length of 12-21 nucleotide units.
 57. The gapmeroligonucleotide of claim 44, wherein the gapmer oligonucleotide has alength of 12-21 nucleotide units.
 58. The gapmer oligonucleotide ofclaim 43, wherein the gapmer oligonucleotide has a length of 15-17nucleotide units.
 59. The gapmer oligonucleotide of claim 44, whereinthe gapmer oligonucleotide has a length of 15-17 nucleotide units. 60.The gapmer oligonucleotide of claim 43, wherein the gapmeroligonucleotide has a length of 13, 14, 15, 16, 17 or 18 nucleotideunits.
 61. The gapmer oligonucleotide of claim 44, wherein the gapmeroligonucleotide has a length of 13, 14, 15, 16, 17 or 18 nucleotideunits in length.